Novel intergenic elements for enhancing gene expression

ABSTRACT

The present invention relates to nucleic acid fragments and constructs comprising genomic nucleotide sequences, which are present upstream of Rb1 and p15C that are associated with intergenic transcription, for the production of a gene product of interest in a eukaryotic, preferably mammalian, host cell in the presence of a stringent selectable marker. The invention further relates to host cells comprising the nucleic acid constructs, to methods for generating the host cells and to methods for producing a gene product of interest using the host cells.

FIELD OF THE INVENTION

The present invention relates to the field of molecular biology and biotechnology. More specifically the present invention relates to means and methods for improving the selection of host cells with high expression levels.

BACKGROUND OF THE INVENTION

Bioactive proteins are produced in various host cells, ranging from bacteria and yeast to mammalian cells. Mammalian cells as host cell are preferred when the protein requires certain posttranslational modifications, such as glycosylation to function properly. In general, proteins produced in mammalian cells are expressed from a so-called ‘transgene’ encoding the protein of interest. To ensure that the right, protein-producing cell is selected, the transgene coding for the gene of interest is coupled to a second transgene encoding a selectable marker that most often is placed on the same vector. When a selection agent is added to the cell culture that has been transfected with the plasmid harboring the transgene, only those cells will survive that also harbor the selectable marker. A common problem is that the stringency of selection is often low. That implies that the cell has to make only very small amounts of selection protein in order to survive the toxic selection agent. In particular when the selection marker is an enzyme that neutralizes the toxic selection agent, these problems occur. One enzyme molecule can neutralize many molecules of selection agent in the course of time. Neomycin and the aminoglycoside phosphotransferase (neomycin) selection marker are an example of such combination. The limited requirement of selection marker protein has also implications for the expression levels of the transgenic protein. Low expression levels of selection marker can, for instance, be achieved by incorporation of only few copies of the plasmid. This, however, implies that also only few gene copies are available for the expression of the transgene protein, with low transgenic protein expression levels as result. Therefore, low expression levels of the protein of interest commonly accompany low selection stringency. This is obviously an unwanted side effect of low selection stringency.

An improvement in selection stringency can be seen when Zeocin and the Zeocin selection marker are used. The Zeocin selection protein is a selection marker protein that does not act as an enzyme. It stoichiometrically binds two Zeocin selection molecules and does not further process these molecules. Thus the available Zeocin selection proteins have only a limited capacity to neutralize a certain number of Zeocin molecules added to the culture medium. Therefore, the cell must produce much more Zeocin than for instance the Neomycin selection marker mRNA to produce enough selection protein to respectively neutralize Zeocin or Neomycin. When coupled to a gene of interest, this commonly also results in higher mRNA levels that encode the gene product of interest. These higher mRNA levels in turn signify higher expression levels of the gene product of interest.

Stably transfected clones can only be selected for the expression levels of the selection marker and not for the expression level of the gene of interest. Because of this, it is preferable that the expression of the gene of interest is directly linked to the expression level of the selection marker. There are multiple ways to physically couple the gene of interest to the gene encoding the selection marker gene. An IRES (Internal Ribosome Entry Site) sequence can be placed between the gene of interest and the gene encoding the selection marker. This creates a bicistronic mRNA from which both the gene product of interest and the selection protein are translated (Rees et al., 1996, Biotechniques 20: 102-110). When a high amount of selection protein, such as Zeocin selection protein is needed for the cell to survive, high levels of this bicistronic mRNA are needed. This in turn implies that high levels of mRNA encoding the gene product of interest are available for translation, and that relatively high expression levels of the gene product of interest are achieved. This principle provides higher selection stringency than when the gene of interest and the gene encoding the selection marker are not coupled through an IRES sequence. This procedure to select cell clones that express relatively high levels of the gene product of interest is an accepted and often employed method (see e.g. WO 03/106684, WO 2006/005718 and WO 2007/096399).

Other means to reach a higher level of selection stringency is to use selectable markers that harbor mutations that attenuate but do not completely destroy the activity of the selection marker. In order to neutralize a similar number of toxic selection molecules in the culture medium more mutated, more impaired selection protein has to be produced than the wild type selection protein. When coupled to the gene of interest through an IRES sequence, the higher impaired selection marker mRNA levels warrant that there is also more mRNA of the gene of interest available for translation. (see e.g. WO 01/32901 and WO 2006/048459)

In yet another example of high selection stringency systems the translation of the selection marker protein is severely impaired. In this example the modified selection marker gene is placed upstream of the gene of interest, not separated by an IRES sequence. In essence, the optimal ATG translation initiation codon of the selection marker is replaced by a less favorable translation initiation codon, such as GTG or TTG. In either case the translation machinery will not initiate translation on the GTG or even less so on the TTG, but will proceed scanning the mRNA. Provided there are no ATGs present in the selection gene (these have to be removed), the first ATG that will be encountered is the ATG of the gene of interest. In this configuration, high levels of this mRNA have to be produced to obtain enough selection protein, which in turn is needed for the cell to survive. However, these high mRNA levels also warrant that concomitantly high levels of the coupled gene of interest will be translated. Through this principle a system of high selection stringency has been created that results in a) only few colonies that survive the selection procedure and b) these colonies display relatively high expression levels of the gene product of interest. In particular a configuration that couples a TTG Zeocin selection marker to the gene of interest provides extremely high selection pressure. Collectively, these selection systems have been termed STAR-Select (WO 2006/048459 and WO 2007/096399).

The present invention discloses further improved means and methods for high stringency selection of mammalian cells to achieve high expression levels of gene products of interest.

DESCRIPTION OF THE INVENTION Definitions

A “nucleic acid construct” is herein understood to mean a man-made nucleic acid molecule resulting from the use of recombinant DNA technology. A nucleic acid construct is a nucleic acid molecule, either single- or double-stranded, which has been modified to contain segments of nucleic acids, which are combined and juxtaposed in a manner, which would not otherwise exist in nature. A nucleic acid construct usually is a “vector”, i.e. a nucleic acid molecule which is used to deliver exogenously created DNA into a host cell. Common types of vectors may be derived from naturally occurring plasmids, phages and viruses. Vectors usually comprise further genetic elements to facilitate their use in molecular cloning, such as e.g. selectable markers, multiple cloning sites and replication origins functional in one or more host cells and the like.

The term “expression” is typically used to refer to the production of a specific nucleic acid product (preferably a specific RNA product) or a specific protein or proteins, in a cell. In the case of RNA products, it refers to the process of transcription. In the case of proteins, it refers to the processes of transcription, translation and optionally post-translational modifications. In the case of secreted proteins, it refers to the processes of transcription, translation, and optionally post-translational modification (e.g., glycosylation, disulfide bond formation, etc.), followed by secretion. In the case of multimeric proteins, it optionally includes assembly of the multimeric structure from the polypeptide monomers.

One type of nucleic acid construct is an “expression construct” or “expression cassette” or “expression vector”. These terms refer to nucleotide sequences that are capable of effecting expression of a gene in host cells or host organisms compatible with such sequences. Expression constructs, expression cassettes or expression vectors typically include at least suitable transcription regulatory sequences and optionally, 3′ transcription termination signals. Additional factors necessary or helpful in effecting expression may also be present, such as expression enhancer elements.

The term “monocistronic gene” is defined as a gene capable of providing a RNA molecule that encodes one gene product. A “multicistronic transcription unit”, also referred to as multicistronic gene, is defined as a gene capable of providing an RNA molecule that encodes at least two gene products. The term “bicistronic gene”, also referred to as “dicistronic gene”, is defined as a gene capable of providing a RNA molecule that encodes two gene products. A bicistronic gene is therefore encompassed within the definition of a multicistronic gene.

The term peptide herein refers to any molecule comprising a chain of amino acids that are linked in peptide bonds. The term peptide thus includes oligopeptides, polypeptides and proteins, including multimeric proteins, without reference to a specific mode of action, size, 3-dimensional structure or origin. A “polypeptide” as used herein usually comprises at least five amino acids linked by peptide bonds. The terms “protein” or “polypeptide” are used interchangeably. A “fragment” or “portion” of a protein may thus still be referred to as a “protein”. An “isolated protein” is used to refer to a protein which is no longer in its natural environment, for example in vitro or in a recombinant (fungal or plant) host cell. The term peptide also includes post-translational modifications of peptides, e.g. glycosylations, acetylations, phosphorylations, and the like.

A “gene product” of interest or a “transcription unit” as used in the present invention can comprise chromosomal DNA, cDNA, artificial DNA, combinations thereof, and the like. A “gene product of interest” can be any gene product, such as for example a protein, a RNAi, shRNA and the like. Non-limiting examples of a protein of interest are enzymes, immunoglobulin chains, therapeutic proteins like anti-cancer proteins or diagnostic proteins. Transcription units comprising several cistrons are transcribed as a single mRNA.

As used herein, the term “operably linked” refers to a linkage of polynucleotide (or polypeptide) elements in a functional relationship. A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a transcription regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein encoding regions, contiguous and in reading frame.

“Expression control sequence” refers to a nucleic acid sequence that regulates the expression of a nucleotide sequence to which it is operably linked. An expression control sequence is “operably linked” to a nucleotide sequence when the expression control sequence controls and regulates the transcription and/or the translation of the nucleotide sequence. Thus, an expression control sequence can include promoters, enhancers, internal ribosome entry sites (IRES), transcription terminators, a start codon in front of a protein-encoding gene, splicing signal for introns, and stop codons. The term “expression control sequence” is intended to include, at a minimum, a sequence whose presence is designed to influence expression, and can also include additional advantageous components. For example, leader sequences and fusion partner sequences are expression control sequences. The term can also include the design of the nucleic acid sequence such that undesirable, potential initiation codons in and out of frame, are removed from the sequence. It can also include the design of the nucleic acid sequence such that undesirable potential splice sites are removed. It includes sequences or polyadenylation sequences (pA) which direct the addition of a polyA tail, i.e., a string of adenine residues at the 3′-end of a mRNA, sequences referred to as polyA sequences. It also can be designed to enhance mRNA stability. Expression control sequences which affect the transcription and translation stability, e.g., promoters, as well as sequences which effect the translation, e.g., Kozak sequences, are known in eukaryotic (host) cells.

As used herein, the term “promoter” or “transcription regulatory sequence” refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A “constitutive” promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An “inducible” promoter is a promoter that is physiologically or developmentally regulated, e.g. by the application of a chemical inducer. A “tissue specific” promoter is only active in specific types of tissues or cells.

As used herein, an “internal ribosome entry site” or “IRES” refers to an element that promotes direct internal ribosome entry to the translation initiation codon (also known as start codon) of a cistron (a protein encoding region), thereby leading to the cap-independent translation of the gene. See, e.g., Jackson R J, Howe ll M T, Kaminski A (1990) Trends Biochem Sci 15 (12): 477-83) and Jackson R J and Kaminski, A. (1995) RNA 1 (10): 985-1000. The present invention encompasses the use of any cap-independent translation initiation sequence, in particular any IRES element that is able to promote direct internal ribosome entry to the initiation codon of a cistron. “Under translational control of an IRES” as used herein means that translation is associated with the IRES and proceeds in a cap-independent manner. As used herein, the term “IRES” encompasses functional variations of IRES sequences as long as the variation is able to promote direct internal ribosome entry to the initiation codon of a cistron.

As used herein, “cistron” refers to a segment of a polynucleotide sequence (DNA) that contains all the information for production of single polypeptide chain.

Sequence identity is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. “Similarity” between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. “Identity” and “similarity” can be readily calculated by known methods. The terms “sequence identity” or “sequence similarity” means that two (poly)peptide or two nucleotide sequences, when optimally aligned, preferably over the entire length (of at least the shortest sequence in the comparison) and maximizing the number of matches and minimizes the number of gaps such as by the programs ClustalW (1.83), GAP or BESTFIT using default parameters, share at least a certain percentage of sequence identity as defined elsewhere herein. GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizes the number of gaps. Generally, the GAP default parameters are used, with a gap creation penalty=50 (nucleotides)/8 (proteins) and gap extension penalty=3 (nucleotides)/2 (proteins). For nucleotides the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). A preferred multiple alignment program for aligning protein sequences of the invention is ClustalW (1.83) using a blosum matrix and default settings (Gap opening penalty:10; Gap extension penalty: 0.05). Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif. 92121-3752 USA, or using open source software, such as the program “needle” (using the global Needleman Wunsch algorithm) or “water” (using the local Smith Waterman algorithm) in EmbossWlN version 2.10.0, using the same parameters as for GAP above, or using the default settings (both for ‘needle’ and for ‘water’ and both for protein and for DNA alignments, the default Gap opening penalty is 10.0 and the default gap extension penalty is 0.5; default scoring matrices are Blossum62 for proteins and DNAFull for DNA). When sequences have a substantially different overall lengths, local alignments, such as those using the Smith Waterman algorithm, are preferred. Alternatively percentage similarity or identity may be determined by searching against public databases, using algorithms such as FASTA, BLAST, etc.

Nucleotide sequences of the invention may also be defined by their capability to hybridize with the specific nucleotide sequences disclosed herein or parts thereof, under moderate, or preferably under stringent hybridization conditions. Stringent hybridization conditions are herein defined as conditions that allow a nucleic acid sequence of at least about 25, preferably about 50 nucleotides, 75 or 100 and most preferably of about 200 or more nucleotides, to hybridize at a temperature of about 65° C. in a solution comprising about 1 M salt, preferably 6×SSC or any other solution having a comparable ionic strength, and washing at 65° C. in a solution comprising about 0.1 M salt, or less, preferably 0.2×SSC or any other solution having a comparable ionic strength. Preferably, the hybridization is performed overnight, i.e. at least for 10 hours and preferably washing is performed for at least one hour with at least two changes of the washing solution. These conditions will usually allow the specific hybridization of sequences having about 90% or more sequence identity.

Moderate conditions are herein defined as conditions that allow a nucleic acid sequences of at least 50 nucleotides, preferably of about 200 or more nucleotides, to hybridize at a temperature of about 45° C. in a solution comprising about 1 M salt, preferably 6×SSC or any other solution having a comparable ionic strength, and washing at room temperature in a solution comprising about 1 M salt, preferably 6×SSC or any other solution having a comparable ionic strength. Preferably, the hybridization is performed overnight, i.e. at least for 10 hours, and preferably washing is performed for at least one hour with at least two changes of the washing solution. These conditions will usually allow the specific hybridization of sequences having up to 50% sequence identity. The person skilled in the art will be able to modify these hybridization conditions in order to specifically identify sequences varying in identity between 50% and 90%.

The adaptiveness of a nucleotide sequence encoding a gene product of interest to the codon usage of a host cell may be expressed as codon adaptation index (CAI). The codon adaptation index is herein defined as a measurement of the relative adaptiveness of the codon usage of a gene towards the codon usage of highly expressed genes in a particular host cell or organism. The relative adaptiveness (w) of each codon is the ratio of the usage of each codon, to that of the most abundant codon for the same amino acid. The CAI index is defined as the geometric mean of these relative adaptiveness values. Non-synonymous codons and termination codons (dependent on genetic code) are excluded. CAI values range from 0 to 1, with higher values indicating a higher proportion of the most abundant codons (see Sharp and Li, 1987, Nucleic Acids Research 15: 1281-1295; also see: Jansen et al., 2003, Nucleic Acids Res. 31(8):2242-51).

A preferred nucleic acid according to the invention is a nucleic acid construct, wherein the nucleotide sequence encoding the antigen-binding protein is operably linked to a promoter and optionally other regulatory elements such as e.g. terminators, enhancers, polyadenylation signals, signal sequences for secretion and the like. Such nucleic acid constructs are particularly useful for the production of the antigen-binding proteins of the invention using recombinant techniques in which a nucleotide sequence encoding the antigen-binding protein of interest is expressed in suitable host cells such as described in Ausubel et al., “Current Protocols in Molecular Biology”, Greene Publishing and Wiley-Interscience, New York (1987) and in Sambrook and Russell (2001) “Molecular Cloning: A Laboratory Manual (3^(rd) edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York). As used herein, the term “operably linked” refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors found that particular nucleotide sequences that are present several kilobases upstream of (i.e. 5′ to the) the retinoblastoma 1 (Rb1) coding sequence (e.g. SEQ ID NO's: 1-4) and parts thereof as further defined herein) and upstream of (i.e. 5′ to the) the Cyclin-dependent kinase 4 inhibitor B coding sequence (also known as p15; CDKN2B; INK4B; MTS2; TP15) (e.g. SEQ ID NO: 8 and parts thereof as further defined below) when placed in an expression vector (comprising, operably linked, a promoter, a nucleotide sequence encoding a selectable marker functional in a eukaryotic host cell and optionally an open reading frame encoding a gene product of interest) are capable of increasing the number of colonies that are formed under selection conditions, preferably stringent selection conditions, as compared to the same expression vector without these particular sequences under stringent selection conditions. The nucleic acid sequences of the invention were not found to possess any promoter activity, nor are they enhancers, or do they influence transcription of endogenous Rb1 and p15 promoters in trans. The nucleic acid sequences of the invention also do not contain STAR activity. Rather, the nucleic acid sequences of the invention were found to be a source for intergenic transcription. The phenomenon of intergenic transcripts has been discovered in for instance the β-globin locus control locus (LCR) (Ashe et al (1997) Genes Dev. 11:2494-2509). For instance, in fission yeast, transcription of a non-coding RNA upstream of the fbp+ locus was shown to be necessary for expression of fbp+ (Hirota et al. (2008) Nature 456:130-134). Here, transcription through the fbp+ gene resulted in a progressively more open chromatin configuration. Intergenic transcription is often associated with promoter activity, however it is not yet clear whether it may be a cause or a consequence (Preker et al. (2008) Science 322:1851-1854). Without wishing to be bound to any theory, it is thought that intergenic transcripts (low-level and often very unstable intergenic transcripts) are involved in opening up a genomic locus or that the chromatin of the locus is kept open for transcription. Although it is not known whether intergenic transcription is causal for opening chromatin structure or the result of already open and transcribed loci, the phenomenon is considered an important epigenetic hallmark of open chromatin regions in which transcription takes place.

A nucleic acid construct according to the invention can be used to select eukaryotic cells, preferably plant cells or mammalian cells, that have high expression levels of a gene product of interest, by selecting for the expression of the selectable marker. Subsequently or simultaneously, one or more of the selected cell(s) can be identified, and further used for expression of high levels of the gene product of interest.

The present invention is based on an impaired efficiency of expression of a selectable marker. Expression of a selectable marker can be detected using routine methods known to the person skilled in the art, e.g. by determining the number of surviving colonies after a normal selection period. As is well known to the person skilled in the art there are a number of parameters that indicate the expression level of a selection marker polypeptide such as, the maximum concentration of selection agent to which cells are still resistant, number of surviving colonies at a given concentration, growth speed (doubling time) of the cells in the presence of selection agent, combinations of the above, and the like. By using the present invention, cells can be identified that have high levels of expression of the selectable marker.

In a first aspect, the present invention relates to a nucleic acid fragment comprising or consisting of: a) between 1,000 and 15,000 consecutive nucleotides of a genomic region that is present upstream of the translation initiation site of a vertebrate Rb1 gene; or, b) at least 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2250, 2500, 2750, 3000 or 3500 consecutive nucleotides from a genomic region that is present from 10.5 to 7 kilobases upstream of the translation initiation site of a vertebrate p15 gene; wherein the fragment, when directly flanking an expression cassette having the nucleotide sequence of SEQ ID NO: 9 both up- and downstream of the expression cassette, produces at least 50, 75, 90, 100, 101, 110, 125 or 150% of number of colonies obtained with the same expression cassette when flanked with STARs 7 and 67 upstream of the expression cassette and STAR 7 downstream of the expression cassette (SEQ ID NO: 10), when tested under the conditions of Example 1. Preferably the fragment has at least 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% nucleotide sequence identity over its entire length with at least 1000, 1500, 2000, 3000, 4000, 5000, 6000, or all of the consecutive nucleotides of at least one of SEQ ID NO's: 1-4 or 8. In a preferred embodiment the nucleic acid fragment is a fragment which has at least 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% nucleotide sequence identity over its entire length with at least 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2250, 2500, 2750, 3000 or 3500 consecutive nucleotides from SEQ ID NO's: 1-4 or 8.

The nucleic acid fragment preferably is an isolated nucleic acid fragment, which is understood to mean a fragment isolated or purified from its natural environment. Preferably, the nucleic acid fragment is from a mammalian genome, more preferably from a primate or rodent genome, and most preferably the nucleic acid fragment is from a human, mouse, rat, hamster, bovine, chicken, dog, cavia, pig or rabbit genome. Preferred nucleic acid fragment are from SEQ ID NO's: 1 or 8 (human), SEQ ID NO: 2 (mouse), SEQ ID NO: 3 (bovine) or SEQ ID NO: 4 (cavia).

In a further preferred embodiment the nucleic acid fragment is selected from the group consisting of fragments having at least 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% nucleotide sequence identity over their entire length with a fragment comprising or consisting of nucleotide residues 1-1019, 1-1482, 1-2018, 1-3498, 479-2018 or 479-1482 of SEQ ID NO: 5, nucleotide residues 1-2448, 1-3424 or 2425-3424 of SEQ ID NO: 6, nucleotide residues 1-3064, 1-2500 or 1-2000 of SEQ ID NO: 7 and nucleotide residues 1-1500, 822-3352 or 1-3352 of SEQ ID NO: 8. More preferably, the nucleic acid fragment is selected from the group consisting of fragments having at least 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% nucleotide sequence identity over their entire length with a fragment comprising or consisting of nucleotide residues 1-3498, 1-2018 or 1-1482 of SEQ ID NO: 5, nucleotide residues 1-3424 or 2425-3424 of SEQ ID NO: 6, nucleotide residues 1-2500 or 1-3064 of SEQ ID NO: 8 and nucleotide residues 822-3352 or 1-3352 of SEQ ID NO: 8. Again more preferably, the nucleic acid fragment is selected from the group consisting of fragments having at least 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% nucleotide sequence identity over their entire length with a fragment comprising or consisting of nucleotide residues 1-2000 of SEQ ID NO: 5; nucleotide residues 2500-3424 of SEQ ID NO: 6; nucleotide residues 1-3064 of SEQ ID NO: 7; and nucleotide residues 850-3352 of SEQ ID NO: 8. Most preferably, the nucleic acid sequence is selected from the group consisting of fragments having at least 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% nucleotide sequence identity over their entire length with a fragment comprising or consisting of nucleotide residues SEQ ID NO: 7 or residues 850-3352 of SEQ ID NO: 8 It is understood that in the above definitions reference is made to the consecutive nucleotide residues of the indicated SEQ ID NO's.

In a second aspect the invention relates to a nucleic acid construct comprising a nucleic acid fragment as defined above, wherein the fragment is linked to at least one nucleotide that does not naturally occur immediately adjacent to the fragment in the genome from which the fragment is derived. Preferably the nucleic acid construct comprises more than one non-naturally occurring nucleotide attached to the fragment, such as e.g. a stretch of nucleotides comprising one or more restriction sites or adapter sequences that are complementary to PCR primers.

More preferably, the nucleic acid construct comprises a nucleic acid fragment as defined above, wherein the fragment is linked to an expression cassette. The expression cassette preferably comprises at least a promoter operably linked to a nucleotide sequence encoding a gene product of interest. The promoter may be a promoter as defined below. The expression cassette may further comprise a nucleotide sequence encoding a selectable marker functional in a eukaryotic host cell e.g. as described below.

A nucleic acid fragment according to the invention functions ‘in cis’. Hence, it is preferred that in the nucleic acid construct, a nucleic acid fragment of the invention is present within 5 kb, more preferably within 2 kb, still more preferably within 1 kb, most preferably within 500 bp from the expression cassette or more preferably from the most 5′ promoter in the expression cassette. If a nucleic acid fragment of the invention is present downstream of the expression cassette in the construct, the nucleic acid fragment of the invention is present within 5 kb, more preferably within 2 kb, still more preferably within 1 kb, most preferably within 500 bp from the expression cassette or more preferably from the most 3′ transcription terminator sequence and/or polyadenylation site in the expression cassette. Thus, a nucleic acid construct may comprise a nucleic acid fragment of the invention either downstream or upstream of an expression cassette. If in the nucleic acid construct sequence, the nucleic acid fragment of the invention is located downstream of the expression cassette, it is preferred that the nucleic acid fragment is a nucleic acid fragment from upstream of Rb1 as defined above, since these fragment yield more colonies at this position and under stringent conditions as compared to the sequences as defined above that are based on SEQ ID NO: 8, i.e. p15 upstream sequences.

However, in a preferred embodiment a nucleic acid construct comprises a nucleic acid fragment according to the invention both upstream and downstream of the expression cassette. In the nucleic acid construct the nucleic acid fragments according to the invention that are present up- and downstream of the expression cassette may be independently selected from the nucleic acid fragments as defined above. Thus, in the nucleic acid construct, the nucleic acid fragment upstream of the expression cassette may be different from the nucleic acid fragment downstream of the expression cassette. Alternatively, in the nucleic acid construct, the nucleic acid fragments up- and downstream of the expression cassette may be (essentially) identical. Preferably, the configuration of the nucleic acid construct is such that, when in linear form and going from 5′ to 3′ end, the nucleic acid construct comprises the following sequence elements in the following order: a first nucleic acid fragment according to the invention, an expression cassette and a second nucleic acid fragment according to the invention, whereby the expression cassette comprises a transcription unit comprising a promoter operably linked to nucleotide sequence encoding a gene product of interest and optionally a selectable marker. The advantage of an expression cassette being flanked by two nucleic acid fragments of the invention is that a higher number of colonies is obtained when cultured in cells under stringent selection conditions and that expression of the selectable marker and thus also of the gene product of interest is higher as compared to a nucleic acid construct with only one nucleic acid fragment according to the invention.

A “expression cassette” as used herein is a nucleotide sequence comprising at least a promoter functionally linked to a nucleotide sequence encoding a gene product of interest, of which expression is desired. Preferably, the expression cassette further contains transcription termination and polyadenylation sequences. Other regulatory sequences such as enhancers may also be included in the expression cassette. In addition to the nucleotide sequence encoding a gene product of interest, the expression cassette preferably also comprises a nucleotide sequence encoding a selectable marker for selection of host cells comprising the expression cassette. In a preferred embodiment, the nucleotide sequence encoding the gene product of interest and the nucleotide sequence encoding a selectable marker are part of the same (multicistronic) transcription unit in the expression cassette. Hence, the invention provides for an expression cassette preferably comprising in a 5′ to 3′ direction, and operably linked: a) 5′—a promoter—a nucleotide sequence encoding a selectable marker—an open reading frame encoding a gene product of interest—optionally, transcription termination and/or polyadenylation sequences—3′, or b) 5′—a promoter—an open reading frame encoding a gene product of interest—a nucleotide sequence encoding a selectable marker—optionally, transcription termination and/or polyadenylation sequences—3′. The promoter, as well as the other regulatory sequences, must be capable of functioning in the eukaryotic host cell in question, i.e. they must be capable of driving transcription of the gene product of interest and the selectable marker. The promoter is thus operably linked to the transcription unit(s) comprising the selectable marker and the open reading frame encoding a gene product of interest. The expression cassette may optionally further contain other elements known in the art, e.g. splice sites to comprise introns, and the like. In some embodiments, an intron is present behind the promoter and before the sequence encoding an open reading frame.

In other embodiments, an IRES may be present in the transcription unit that contains the selectable marker coding sequence and the sequence encoding the gene product of interest, which IRES may be present in between the open reading frames of the selectable marker and the gene product of interest. Internal ribosome binding site (IRES) elements are known from viral and mammalian genes (Martinez-Salas, 1999, Curr Opin Biotechnol 10: 458-464), and have also been identified in screens of small synthetic oligonucleotides (Venkatesan & Dasgupta, 2001 Mol Cell Biol 21: 2826-2837). The IRES from the encephalomyocarditis virus has been analyzed in detail (Mizuguchi et al., 2000, Mol Ther 1: 376-382). An IRES is an element encoded in DNA that results in a structure in the transcribed RNA at which eukaryotic ribosomes can bind and initiate translation. An IRES permits two or more proteins to be produced from a single RNA molecule (the first protein is translated by ribosomes that bind the RNA at the cap structure of its 5′ terminus, (Martinez-Salas, 1999, supra). Thus, the invention provides an expression cassette preferably comprising in a 5′ to 3′ direction: 5′—a promoter—an open reading frame encoding a gene product of interest—an IRES—a selectable marker—optionally, transcription termination and/or polyadenylation sequences—3′ or 5′-promoter—a selectable marker—an IRES—an open reading frame encoding a gene product of interest—optionally, transcription termination and/or polyadenylation sequences—3′. A promoter to be applied in the expression cassettes comprised in the nucleic acid constructs of the invention preferably is functional in a eukaryotic host cell, more preferably, the promoter is functional in a plant or animal host cell, still more preferably the promoter is functional in a vertebrate host cell and most preferably in a mammalian host cell, for initiating transcription of the transcription unit. Promoters can be constitutive or regulated, and can be obtained from various sources, including viruses, prokaryotic, or eukaryotic sources, or artificially designed. Expression of nucleic acids of interest may be from the natural promoter or derivative thereof or from an entirely heterologous promoter (Kaufman, 2000, Mol. Biotechnol 16: 151-160). According to the present invention, strong promoters that give high transcription levels in the eukaryotic cells of choice are preferred. Some well-known and frequently used strong promoters for expression in eukaryotic cells comprise promoters derived from viruses, such as adenovirus, e.g. the ElA promoter, promoters derived from cytomegalovirus (CMV), such as the CMV immediate early (IE) promoter (referred to herein as the CMV promoter) (obtainable e.g. from pcDNA, Invitrogen), promoters derived from Simian Virus 40 (SV40) (Das et al, 1985, Prog Nucleic Acid Res Mol Biol. 32: 217-36), and the like. Suitable strong promoters can also be derived from eukaryotic cells, such as methallothionein (MT) promoters, an elongation factor (EF-1α) promoter, an ubiquitin C or UB6 promoter (Gill et al., 2001, Gene Therapy 8: 1539-1546; Schorpp et al, 1996, Nucleic Acids Res 24: 1787-8), an actin promoter such as a β-actin promoter, e.g. a hamster or human β-actin promoter (SEQ ID NO: 11), an immunoglobulin promoter, a heat shock promoter and the like. Testing for promoter function and strength of a promoter is a matter of routine for a person skilled in the art, and in general may for instance encompass cloning a reporter gene such as lacZ, luciferase, GFP, etc. behind the promoter sequence, and test for expression of the reporter gene. Of course, promoters may be altered by deletion, addition, mutation of sequences therein, and tested for functionality, to find new, attenuated, or improved promoter sequences. Preferred promoters for use in the present invention are a human β-actin promoter, a CMV promoter, an SV40 promoter, an ubiquitin C promoter or an EF1-alpha promoter.

An open reading frame is herein understood as a nucleotide sequence comprising in a 5′ to 3′ direction 1) a translation initiation codon, 2) one or more codons coding for a gene product of interest, preferably a protein, and 3) a translation stop codon, whereby it is understood that 1), 2) and 3) are operably linked in frame. The open reading frame will thus consist of a multiple of 3 nucleotides (triplets).

A gene product of interest according to the invention can be any gene product, e.g. a protein. A gene product of interest may be a monomeric protein or a (part of a) multimeric protein. A multimeric protein comprises at least two polypeptide chains. Non-limiting examples of a protein of interest according to the invention are enzymes, hormones, immunoglobulins or chains or fragments thereof, therapeutic proteins like anti-cancer proteins, blood coagulation proteins such as Factor VIII, multi-functional proteins, such as erythropoietin, diagnostic proteins, or proteins or fragments thereof useful for vaccination purposes, all known to the person skilled in the art.

A gene product of interest may be from any source, and in certain embodiments is a mammalian protein, an artificial protein (e.g. a fusion protein or mutated protein), and preferably is a human protein.

In a preferred embodiment, a nucleotide sequence encoding a gene product of interest is codon optimized for the host cell in which the peptide of interest is to be expressed, using the codon adaptation index of the host cell. The adaptiveness of a nucleotide sequence encoding an enzyme to the codon usage of a host cell may be expressed as codon adaptation index (CAI). The codon adaptation index is herein defined as a measurement of the relative adaptiveness of the codon usage of a gene towards the codon usage of highly expressed genes in a particular host cell or organism. The relative adaptiveness (w) of each codon is the ratio of the usage of each codon, to that of the most abundant codon for the same amino acid. The CAI index is defined as the geometric mean of these relative adaptiveness values. Non-synonymous codons and termination codons (dependent on genetic code) are excluded. CAI values range from 0 to 1, with higher values indicating a higher proportion of the most abundant codons (see Sharp and Li, 1987, Nucleic Acids Research 15: 1281-1295; also see: Kim et al., Gene. 1997, 199:293-301; zur Megede et al., Journal of Virology, 2000, 74: 2628-2635). Preferably, a nucleotide sequence encoding a gene product of interest has a CAI of at least 0.5, 0.6, 0.7, 0.8, 0.9 or 0.95.

In one embodiment, a nucleic acid construct of the present invention is used when the ultimate goal is not the production of a polypeptide of interest, but rather an RNA molecule, e.g. for producing increased quantities of RNA from an expression cassette, which may be used for purposes of regulating other genes (e.g. RNAi, antisense RNA), gene therapy, in vitro protein production, etc.

For the production of multimeric proteins, two or more nucleic acid constructs according to the invention can be used. For example, both expression cassettes can be multicistronic nucleic acid constructs, each coding for a different selectable marker protein, so that selection for both expression cassettes is possible. This embodiment is advantageous, e.g. for the expression of the heavy and light chain of immunoglobulins such as antibodies. It will be clear that both nucleic acid constructs may be placed on one nucleic acid molecule or both may be present on a separate nucleic acid molecule, before they are introduced into host cells. An advantage of placing them on one nucleic acid molecule is that the two nucleic acid constructs are present in a single predetermined ratio (e.g. 1:1) when introduced into host cells. On the other hand, when present on two different nucleic acid molecules, this allows the possibility to vary the molar ratio of the two nucleic acid constructs when introducing them into host cells, which may be an advantage if the preferred molar ratio is different from 1:1 or when it is unknown beforehand what is the preferred molar ratio, so that variation thereof and empirically finding the optimum can easily be performed by the skilled person. According to the invention, preferably at least one of the nucleic acid constructs, but more preferably each of them, comprises a at least one but preferably two nucleic acid fragments according to the invention.

In another embodiment, the different subunits or parts of a multimeric protein are present in a single expression construct. Useful configurations of anti-repressors combined with expression constructs have been described in WO 2006/048459 (e.g. page 40), incorporated by reference herein.

In a preferred embodiment, the gene product of interest is a coagulation factor such as Factor VIII or factor VII, interferons and interleukins, such as human interferon-gamma or therapeutic, anti-cancer monoclonal antibodies such as Herceptin (anti-EGF receptor) or Avastin (anti-vascular endothelial growth factor (VEGF)) or EPO.

A nucleic acid construct of the invention can be present in the form of double stranded DNA, having with respect to the selectable marker and the open reading frame encoding a gene product of interest a coding strand and a non-coding strand, the coding strand being the strand with the same sequence as the translated RNA, except for the presence of T instead of U. Hence, an AUG startcodon is coded for in the coding strand by an ATG sequence, and the strand containing this ATG sequence corresponding to the AUG startcodon in the RNA is referred to as the coding strand of the DNA. It will be clear to the skilled person that startcodons or translation initiation sequences are in fact present in an RNA molecule, but that these can be considered equally embodied in a DNA molecule coding for such an RNA molecule; hence, wherever the present invention refers to a startcodon or translation initiation sequence, the corresponding DNA molecule having the same sequence as the RNA sequence but for the presence of a T instead of a U in the coding strand of said DNA molecule is meant to be included, and vice versa, except where explicitly specified otherwise. In other words, a startcodon is for instance an AUG sequence in RNA, but the corresponding ATG sequence in the coding strand of the DNA is referred to as startcodon as well in the present invention. The same is used for the reference of ‘in frame’ coding sequences, meaning triplets (3 bases) in the RNA molecule that are translated into an amino acid, but also to be interpreted as the corresponding trinucleotide sequences in the coding strand of the DNA molecule.

A selectable marker to be applied in the expression cassettes comprised in the nucleic acid constructs of the invention preferably is functional in a eukaryotic host cell, more preferably, the marker is functional in a plant or animal host cell, still more preferably in a vertebrate host cell and most preferably in a mammalian host cell.

The term “selectable marker” is a term familiar to one of ordinary skill in the art and is used herein to describe any genetic entity which, when expressed, can be used to select for a cell or cells containing (and/or expressing) the selectable marker. Selectable markers may be dominant or recessive or bidirectional. The selectable marker may be a gene coding for a product which confers to a cell expressing the gene resistance to a selection agent such as e.g. an antibiotic or herbicide. The selectable marker may e.g. encode a selection protein that is able to neutralize or inactivate a toxic selection agent and protects the host cell from the agent's lethal or growth-inhibitory effects. Other selectable markers complement a growth-inhibitory deficiency in the cell under certain conditions. Examples of such genes include a gene which confers prototrophy to an auxotrophic strain. The term “reporter” is mainly used to refer to visible markers, such as green fluorescent protein (GFP), eGFP, luciferase, GUS and the like, as well as nptII markers and the like. Such reporters can be used for selecting cells expressing the visible marker by actively sorting cells expressing the marker from cells that do not, e.g. using a fluorescence activated cell sorter (FACS) for selecting cells that express a fluorescent marker protein. Preferably, the selectable marker according to the invention provides resistance against lethal and/or growth-inhibitory effects of a selection agent.

A nucleotide sequence encoding a selectable marker for use in the present invention encodes a protein that can be used for selection of eukaryotic host cells, e.g. because upon expression of the protein in the host cell it provides a growth advantage to the host cells expressing the selectable marker protein, as compared to host that do not. A preferred nucleotide sequence encoding a selectable marker provides resistance to a selection agent (e.g. an antibiotic) upon expression of the encoded selectable marker protein in the host cell, which selection agent causes lethality and/or growth inhibition of host cells not expressing the selectable marker protein. The selectable marker according to the invention must thus be functional in a eukaryotic host cell, and hence being capable of being selected for in eukaryotic host cells. Any selectable marker polypeptide fulfilling this criterion can in principle be used according to the present invention. Such selectable markers are well known in the art and routinely used when eukaryotic host cell clones are to be obtained, and several examples are provided herein.

For convenience and as generally accepted by the skilled person, in many publications as well as herein, often the gene encoding for the selectable marker and the selectable marker that causes resistance to a selection agent is referred to as the ‘selectable agent (resistance) gene’ or ‘selection agent (resistance) protein’, respectively, although the official names may be different, e.g. the gene coding for the protein conferring resistance to neomycin (as well as to G418 and kanamycin) is often referred to as neomycin (resistance) (or neo') gene, while the official name is aminoglycoside 3′-phosphotransferase gene.

In a preferred embodiment of the invention, the selectable marker provides resistance against lethal or growth-inhibitory effects of a selection agent selected from the group consisting of the bleomycin family of antibiotics, puromycin, blasticidin, hygromycin, an aminoglycoside antibiotic, methotrexate, and methionine sulphoximine.

A nucleotide sequence encoding a selectable marker providing resistance to bleomycin family of antibiotics is e.g. a nucleotide sequence encoding a wild-type “ble” gene, including but not limited to Sh ble, Tn5 ble and Sa ble or a variant thereof. An example thereof is depicted in SEQ ID NO: 14. In general the gene products encoded by the ble genes confer to their host resistance to the copper-chelating glycopeptide antibiotics of the bleomycin family, which are DNA-cleaving glycopeptides. Examples of antibiotics of the bleomycin family for use as selection agents in accordance with the present invention include but are not limited to bleomycin, phleomycin, tallysomycin, pepleomycin and Zeocin™. Zeocin is particularly advantageous as a selection agent, because the zeocin-resistance protein (zeocin-R) acts by binding the drug and thereby rendering it harmless. Therefore it is easy to titrate the amount of drug that kills cells with low levels of zeocin-R expression, while allowing the high-expressors to survive. Most if not all other antibiotic-resistance selectable markers in common use are enzymes, and thus act catalytically (i.e. not in a given, e.g. 1:1, stoichiometry with the selection agent). Hence, the antibiotic zeocin is a preferred selectable marker.

A nucleotide sequence encoding a selectable marker providing resistance to the aminoglycoside antibiotic is e.g. a nucleotide sequence encoding a wild-type aminoglycoside 3′-phosphotransferase or a variant thereof. An aminoglycoside according to the present invention are the commonly known aminoglycoside antibiotics (Mingeot-Leclercq, M. et al., 1999, Chemother. 43: 727-737) comprising at least one amino-pyranose or amino-furanose moiety linked via a glycosidic bond to the other half of the molecule. Their antibiotic effect is based on inhibition of protein synthesis. Examples of aminoglycoside antibiotics for use as selection agents in accordance with the present invention include but are not limited Kanamycin, Streptomycin, Gentamicin, Tobramycin, G418 (Geneticin), Neomycin B (Framycetin), Sisomicin, Amikacin, Isepamicin and the like.

Other examples of selectable markers which can be used in the invention are DHFR, cystathionine gamma-lyase and glutamine synthetase (GS) genes. A potential advantage of the use of these types of metabolic enzymes as selectable marker polypeptides is that they can be used to keep the host cells under continuous selection, which may advantageous under certain circumstances.

The DHFR gene, which can be selected for by methotrexate, especially by increasing the concentration of methotrexate cells can be selected for increased copy numbers of the DHFR gene. The DHFR gene may also be used to complement a DHFR-deficiency, e.g. in CHO cells that have a DHFR⁻ phenotype, in a culture medium with folate and lacking glycine, hypoxanthine and thymidine. If the selectable marker is DHFR, the host cell in advantageous embodiments is cultured in a culture medium that contains folate and which culture medium is essentially devoid of hypoxanthine and thymidine, and preferably also of glycine. In general, with “culture medium is essentially devoid” is meant herein that the culture medium has insufficient of the indicated component present to sustain growth of the cells in the culture medium, so that a good selection is possible when the genetic information for the indicated enzyme is expressed in the cells and the indicated precursor component is present in the culture medium. Preferably, the indicated component is absent from the culture medium. A culture medium lacking the indicated component can be prepared according to standard methods by the skilled person or can be obtained from commercial media suppliers.

Selection for a glutamine synthetase (GS) gene, e.g. a wild-type human or mouse glutamine synthetase gene, is possible in cells having insufficient GS (e.g. NS-O cells) by culturing in media without glutamine, or alternatively in cells having sufficient GS (e.g. CHO cells) by adding an inhibitor of GS, methionine sulphoximine (MSX).

Cystathionine gamma-lyase (EC 4.4.1.1) is an enzyme that is crucial for the synthesis of the amino acid L-cysteine. CHO cells are natural auxotrophs for the conversion of cysthathionine to cysteine. Therefore, the cystathionine gamma-lyase (cys-lyase) gene, e.g. from mouse or human, can be used for selection of cells by complementation by culturing cells in media without L-cysteine and L-cystine. Selection on the basis of the cys-lyase marker may require the non-toxic precursor L-cystathionine to be present in the culture medium. The use of cys-lyase as selectable marker in some vertebrate cell lines may first require inactivation (knock-out) of the endogenous cystathionine gamma-lyase genes.

Further selectable markers and their selection agents that could be used in the context of the present invention, are for instance described in Table 1 of U.S. Pat. No. 5,561,053, incorporated by reference herein; see also Kaufman, Methods in Enzymology, 185:537-566 (1990), for a review of these selectable markers and their selection agents.

In a preferred embodiment, the expression cassette in a nucleic acid construct of the present invention, comprises a selectable marker that is a stringent selection marker. A stringent selection marker is herein understood as a selection marker that requires to be transcribed (and/or expressed) at high level in the host cell expressing the marker for that host cell to be selected, i.e. for that host cell to survive the applied selection. In the context of the present invention, the stringency of the selectable marker is preferably increased by at least one of a) reducing the translation (initiation) efficiency of the selectable marker and b) reducing the activity and/or efficacy of the selectable marker polypeptide. Therefore, the expression cassette in a nucleic acid construct of the present invention, preferably comprises a nucleotide sequence encoding the selectable marker which nucleotide sequence is a least one of:

a) a nucleotide sequence having a mutation in the startcodon that decreases the translation initiation efficiency of the selectable marker polypeptide in a eukaryotic host cell;

b) a nucleotide sequence that is part of a multicistronic transcription unit comprising i) the nucleotide sequence encoding the selectable marker; and, ii) a functional open reading frame comprising in a 5′ to 3′ direction a translation initiation codon, at least one amino acid codon and a translation stop codon; wherein the stop codon of functional open reading frame is present between 0 and 250 nucleotides upstream of the separate translation initiation codon of the nucleotide sequence encoding the selectable marker, and wherein the sequence separating the stop codon of functional open reading frame and the separate translation initiation codon of the nucleotide sequence encoding the selectable marker is devoid of translation initiation codons; and,

c) a nucleotide sequence encoding a selectable marker polypeptide comprising a mutation encoding at least one amino acid change that reduces the activity of the selectable marker polypeptide compared to its wild-type counterpart.

Nucleotide sequences encoding a selectable marker having a mutation in the (translation) startcodon (a sub-optimal non-AUG initiation codon) that decreases the translation initiation efficiency of the selectable marker polypeptide in a eukaryotic host cell are known in the art (see e.g. WO 2007/096399). A non-ATG (non-AUG) startcodon is herein understood as a translation initiation codon comprising a mutation in the startcodon that decreases the translation initiation efficiency of the selectable marker polypeptide in a eukaryotic host cell. Examples of non-ATG start codons that may be used for the coding sequence of the selectable marker in the invention include e.g. GTG, TTG, CTG, ATT, and ACG. In a preferred embodiment, the ATG startcodon is mutated into a GTG startcodon. More preferably, the ATG startcodon is mutated to a TTG startcodon, which provides even lower expression levels of the selectable marker polypeptide than with the GTG startcodon. When using a non-ATG startcodon, it is preferred that the non-ATG start codon is present in an optimal context for translation initiation codon, such as a Kozak consensus sequence as herein defined below. When applying a non-ATG startcodon for the selectable marker the nucleotide sequence coding for the selectable marker can be mutated to be devoid of internal ATG codons, particularly devoid of internal ATG codons that are in frame with the non-ATG start codon. This is preferred in constructs wherein the selectable marker is upstream of a nucleotide sequence coding for a gene product of interest without using an IRES in between the sequences coding for the gene product of interest and the marker. WO 2006/048459 discloses how to bring this about (e.g. by substitution, insertion or deletion, preferably by substitution) and how to test the resulting selectable marker polypeptides for functionality.

The second option for reducing the efficiency of translation initiation in b) above, uses a (short) functional open reading frame (pp^(x); wherein pp^(x) is a petit peptide of x amino acid residues) directly preceding the translation initiation codon of the selectable marker. The length of the functional open reading frame (pp^(x)) can be varied in order to fine tune low levels of translational efficiency of the selectable marker polypeptide, so that the exact required level of stringency of selection is obtained. Thus, the functional open reading frame may thus encode at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80 or 90 amino acid residues and preferably encodes no more than 200, 180, 160, 150, 140, 130, 120, 110, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, or 90 amino acid residues with a startcodon at the 5′ and a stopcodon at the 3′ end. By thus varying the length of the functional open reading frame (pp^(x)) that immediately precedes the sequence encoding the selectable marker in the transcript, a near continuous range of translational efficiencies of the selectable marker is provided. The functional open reading frame (pp^(x)) may be located immediately upstream of the separate startcodon of the selectable marker, in which case the stopcodon of the functional open reading frame is immediately adjacent to the start codon of the sequence coding for the selectable marker. Alternatively the stopcodon of the upstream functional open reading frame (pp^(x)) and the startcodon of the sequence coding for the selectable marker may be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160 180, 200, 250 or more nucleotides. Variation of the length of the spacer sequence separating the stopcodon of the upstream functional open reading frame (pp^(x)) and the startcodon of the sequence coding for the selectable marker adds a further level of fine tuning of the translational efficiency of the selectable marker. The spacer sequence separating the stop codon of functional open reading frame (pp^(x)) and the separate translation initiation codon of the nucleotide sequence encoding the selectable marker is devoid of translation initiation codons. Preferably therefore, the spacer sequence lacks ATG codons. More preferably, the spacer sequence also lacks suboptimal non-ATG codons such as GTG, TTG, CTG, ATT, and ACG (see below) embedded in a Kozak sequence (see below). Most preferably, the spacer sequence is devoid of any of the ATG, GTG, TTG, CTG, ATT, and ACG codons. In a further preferred embodiment, the spacer sequence separating the stop codon of functional open reading frame (pp^(x)) and the separate translation initiation codon of the nucleotide sequence encoding the selectable marker is devoid of stopcodons, i.e. lacks TAA, TAG and TGA codons.

In a preferred embodiment, at least one of the translation initiation codons of the nucleotide sequence encoding the selectable marker and of the functional open reading frame (pp^(x)) is an ATG codon. More preferably at least the initiation codon of the nucleotide sequence encoding the functional open reading (ppX) is an ATG codon, in which case the initiation codon of the nucleotide sequence encoding the selectable marker can be a non-ATG startcodon (also known as suboptimal or less-favorable translation initiation codon), in order to allow for even more stringent selection (see above). Most preferably both the translation initiation codons of the nucleotide sequence encoding the selectable marker and the functional open reading frame (pp^(x)) are ATG codons. However, the invention does not exclude that the initiation codon of the nucleotide sequence encoding the functional open reading (pp^(x)) is a non-ATG startcodon.

In one embodiment, at least one of the initiation codons of the nucleotide sequence encoding the selectable marker and the functional open reading frame (pp^(x)) is embedded in a Kozak consensus sequence. The Kozak consensus sequence (for vertebrate host cells) is herein defined as ANN(AUG)N (SEQ ID NO: 11) and GNN(AUG)G (SEQ ID NO: 12), wherein (AUG) stands for the initiation codon of the relevant coding sequence. Preferably, both N's preceding the (AUG) are C's. A more preferred Kozak consensus sequence is GCCRCC(AUG)G (SEQ ID NO: 13), wherein R is a purine. In a further preferred embodiment, the Kozak consensus sequence may be preceded by yet another GCC triplet.

A preferred selectable markers preceded by a functional open reading frame (pp^(x)) is e.g. pp⁹⁰ZEO (a pp^(x) open reading frame that encodes 90 amino acids preceding the zeomycin resistance protein; the pp⁹⁰ coding sequence is given in SEQ ID NO: 15).

In one embodiment, alternatively or in combination with a decreased translation initiation efficiency of a) or b) above, it can be beneficial to also provide for decreased translation elongation efficiency of the selectable marker polypeptide. This may be achieved by e.g. mutating the sequence coding the selectable marker polypeptide so as to decrease the adaptation of the codon usage to the host cell in question. This again provides a further level of controlling the stringency of selection of the nucleic acid constructs of the invention. Thus, a nucleotide sequence encoding a selectable marker protein, is preferably adapted to a codon usage to that is suboptimal in host cell in question. An codon adapted nucleotide sequence in accordance with the present invention preferably has a CAI of no more than 0.7, 0.6, 0.5, 0.4, 0.3 or 0.2 (see above for definition of CAI).

In one embodiment, alternatively or in combination with the embodiments of selectable markers with a decreased translation initiation efficiency as described in a) or b) above, mutants or derivatives of selectable markers are suitably used according to the invention, and are therefore included within the scope of the term ‘selectable marker’, as long as the selectable marker is still functional. Mutants or derivatives of a selectable marker preferably have reduced activity of the selectable marker compared to its wild-type counterpart allowing a further level of control in fine tuning of the stringency of selection of the nucleic acid constructs of the invention. Alternatively or in combination with one or more other embodiments, in a preferred embodiment, the nucleotide sequence encoding the selectable marker encodes a selectable marker polypeptide comprising one or more mutations that (collectively) reduce the activity of the selectable marker polypeptide compared to its wild-type counterpart. The activity of the mutated selectable marker polypeptide can be or more than 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 or 1% to its wild-type counterpart.

As non-limiting examples, proline at position 9 in the zeocin resistance polypeptide may be mutated, e.g. to Thr or Phe (see e.g. example 14 of WO 2006/048459, incorporated by reference herein), and for the neomycin resistance polypeptide, amino acid residue 182 or 261 or both may further be mutated (see e.g. WO 01/32901). A preferred selectable marker polypeptide with reduced activity is a zeocin resistance polypeptide having the amino acids sequence of SEQ ID NO: 14 wherein the glutamic acid at position 21 is changed into glycine, and the alanine at position 76 is changed into threonine (Zeo^(EPP5)).

A particularly preferred stringent selectable marker is pp⁸ZEO^(EPP5), which combines a pp^(x) open reading frame of 8 amino acids and the Zeo^(EPP5) zeocin resistance protein with reduced activity. The sequence of pp⁸ZEO^(EPP5) is depicted in SEQ ID NO: 16.

A nucleic acid construct according to the invention is preferably comprised in a plasmid or an expression construct can be a plasmid. A plasmid can easily be manipulated by methods well known to the person skilled in the art, and can for instance be designed for being capable of replication in prokaryotic and/or eukaryotic cells. Alternatively, a nucleic acid construct may be a vector. Many vectors can directly or in the form of isolated desired fragment therefrom be used for transformation of eukaryotic cells and will integrate in whole or in part into the genome of such cells, resulting in stable host cells comprising the desired nucleic acid in their genome.

Conventional expression systems are DNA molecules in the form of a recombinant plasmid or a recombinant viral genome. The plasmid or the viral genome is introduced into (eukaryotic host) cells and preferably integrated into their genomes by methods known in the art, and several aspects hereof have been described in WO 2006/048459 (e.g. pages 30-31), incorporated by reference herein.

In one embodiment, a nucleic acid construct according to the invention comprises an additional selection marker, e.g. a DHFR metabolic selection marker as described supra. An advantage of such a nucleic acid construct is that selection of a host cell with high expression can be established by use of a selection marker operably linked with an IRES, e.g. zeocin, neomycin, etc, whereas after the selection of a host cell with high expression the antibiotic selection is discontinued and either continuous or intermittent selection is done using the additional selection marker. The multicistronic transcription units in this embodiment are at least tricistronic.

It is preferred to use separate nucleic acid constructs for the expression of different gene products of interest, also when these form part of a multimeric protein (see e.g. example 13 of WO 2006/048459, incorporated by reference herein): the heavy and light chain of an antibody each are encoded by a separate transcription unit according to the invention. When two transcription units of the invention are to be selected for according to the invention in a single host cell, each one preferably contains the coding sequence for a different selectable marker, to allow selection for both transcription units. Of course, both transcription units may be present on a single nucleic acid molecule or alternatively each one may be present on a separate nucleic acid molecule.

In a third aspect, the present invention relates to an expression vector or an expression construct comprising a nucleic acid construct according to the invention.

In a fourth aspect, the present invention relates to a host cell, preferably a eukaryotic host cell, comprising a nucleic acid construct according to the invention or an expression vector according to the invention.

The terms “cell” or “host cell” and “cell line” or “host cell line” are respectively defined as a cell and homogeneous populations thereof that can be maintained in cell culture by methods known in the art, and that have the ability to express heterologous or homologous proteins. The host is an eukaryotic host cell such as a cell of fungal, plant, or animal origin. Preferably the host cell is an animal cell of insect or vertebrate origin. More preferably the host cell is a mammalian cell. Preferably, the host cell is a cell of a cell line. Several exemplary host cells that can be used have been described in WO 2006/048459 (e.g. page 41-42), incorporated by reference herein, and such cells include for instance mammalian cells, including but not limited to CHO cells, e.g. CHO-K1, CHO-S, CHO-DG44, CHO-DG44-S, CHO-DUKXBI 1, including CHO cells having a dhfr⁻ phenotype, as well as myeloma cells (e.g. Sp2/0, NSO), HEK 293 cells, HEK 294 cells, and PER.C6 cells. Other examples of host cells that can be used are a U-2 OS osteosarcoma, HuNS-1 myeloma, WERI-Rb-1 retinoblastoma, BHK, Vero, non-secreting mouse myeloma Sp2/0-Ag 14, non-secreting mouse myeloma NSO and NCI-H295R adrenal gland carcinoma cell line.

Such eukaryotic host cells can express desired gene products, and are often used for that purpose. They can be obtained by introduction of a nucleic acid construct of the invention, preferably in the form of an expression construct, an expression cassette or an expression vector according to the invention, into the cells. Preferably, the nucleic acid construct is integrated in the genome of the host cell, which can be in different positions in various host cells, and selection will provide for a clone where the transgene is integrated in a suitable position, leading to a host cell clone with desired properties in terms of expression levels, stability, growth characteristics, and the like.

Alternatively a nucleic acid construct without promoter may be targeted or randomly selected for integration into a chromosomal region that is transcriptionally active, e.g. behind a promoter present in the genome. Selection for cells containing the DNA of the invention can be performed by selecting for the selectable marker polypeptide, using routine methods known by the person skilled in the art. When such a nucleic acid construct without promoter is integrated behind a promoter in the genome, a nucleic acid construct according to the invention can be generated in situ, i.e. within the genome of the host cells.

Preferably the host cells are from a stable clone that can be selected and propagated according to standard procedures known to the person skilled in the art. A culture of such a clone is capable of producing gene product of interest, if the cells comprise the multicistronic transcription unit of the invention.

Introduction of nucleic acid that is to be expressed in a cell, can be done by one of several methods, which as such are known to the person skilled in the art, also dependent on the format of the nucleic acid to be introduced. Said methods include but are not limited to transfection, infection, injection, transformation, and the like. Suitable host cells that express the gene product of interest can be obtained by selection.

In preferred embodiments, a nucleic acid construct according to the invention is integrated into the genome of the eukaryotic host cell according to the invention. This will provide for stable inheritance of the nucleic acid construct.

In a fifth aspect, the present invention relates to a method of generating a host cell for expression of a gene product of interest, wherein the method comprises the steps of: a) introducing into a plurality of host cells a nucleic acid construct according to the invention or a expression vector according to the invention; b) culturing the plurality of host cells obtained in a) under conditions selecting for expression of the selectable marker polypeptide; and, c) selecting at least one host cell expressing the selectable marker polypeptide for expression of the gene product of interest.

Advantages of this method are similar to those described for the method disclosed in WO 2006/048459 (e.g. page 46-47), incorporated by reference herein. While clones having relatively low copy numbers of the nucleic acid construct and high expression levels can be obtained, the selection system of the invention nevertheless can be combined with amplification methods to even further improve expression levels. This can for instance be accomplished by amplification of a co-integrated DHFR gene using methotrexate, for instance by placing DHFR on the same nucleic acid molecule as the multicistronic transcription unit of the invention, or by cotransfection when DHFR is on a separate DNA molecule. The DHFR gene can also be part of a nucleic acid construct of the invention or of the expression vector of the invention.

Selection for the presence of the selectable marker polypeptide, and hence for expression, can be performed during the initial obtaining of the host cell. In certain embodiments, the selection agent is present in the culture medium at least part of the time during the culturing, either in sufficient concentrations to select for cells expressing the selectable marker or in lower concentrations.

In a sixth aspect, the present invention relates to a method of expressing a gene product of interest, comprising culturing a host cell comprising a nucleic acid construct according to the invention or a vector according to the invention, a host cell according to the invention or a host cell obtained in a method according to the invention, and expressing the gene product of interest from the nucleic acid construct. In preferred embodiments, selection agent is no longer present in the culture medium during final the production phase of gene product of interest so as to avoid any risk of contamination of the gene product with trace of the possibly noxious selection agent.

In certain embodiments, an expression vector of the invention encodes an immunoglobulin heavy or light chain or an antigen binding part, derivative and/or analogue thereof. In a preferred embodiment a protein expression unit according to the invention is provided, wherein said protein of interest is an immunoglobulin heavy chain. In yet another preferred embodiment a protein expression unit according to the invention is provided, wherein said gene product of interest is an immunoglobulin light chain. When these two protein expression units are present within the same (host) cell a multimeric protein and more specifically an immunoglobulin, is assembled. Hence, in certain embodiments, the protein of interest is an immunoglobulin, such as an antibody, which is a multimeric protein. Preferably, such an antibody is a human or humanized antibody. In certain embodiments thereof, it is an IgG, IgA, or IgM antibody. An immunoglobulin may be encoded by the heavy and light chains on different expression vectors, or on a single expression vector. Thus, the heavy and light chain can each be present on a separate expression vector, each having its own promoter (which may be the same or different for the two expression vectors), each comprising a transcription unit according to the invention, the heavy and light chain being the gene product of interest, and preferably each coding for a different selectable marker protein, so that selection for both heavy and light chain expression vector can be performed when the expression vectors are introduced and/or present in a eukaryotic host cell. Alternatively, the heavy and light chain coding sequences can be present on a single expression vector comprising a multicistronic transcription unit according to the invention, driven from a single promoter, and wherein the light and heavy chains are the gene products of interest with an IRES in between their respective coding sequences.

Culturing a cell is done to enable it to metabolize, and/or grow and/or divide and/or produce gene products of interest. This can be accomplished by methods well known to persons skilled in the art, and includes but is not limited to providing nutrients for the cell. The methods comprise growth adhering to surfaces, growth in suspension, or combinations thereof. Culturing can be done for instance in dishes, roller bottles or in bioreactors, using batch, fed-batch, continuous systems such as perfusion systems, and the like. In order to achieve large scale (continuous) production of recombinant gene products through cell culture it is preferred in the art to have cells capable of growing in suspension, and it is preferred to have cells capable of being cultured in the absence of animal- or human-derived serum or animal- or human-derived serum components.

The conditions for growing or multiplying cells (see e.g. Tissue Culture, Academic Press, Kruse and Paterson, editors (1973)) and the conditions for expression of the recombinant product are known to the person skilled in the art. In general, principles, protocols, and practical techniques for maximizing the productivity of mammalian cell cultures can be found in Mammalian Cell Biotechnology: a Practical Approach (M. Butler, ed., IRL Press, 1991).

In a preferred embodiment, a method of expressing a gene product of interest according to the invention further comprises harvesting the gene product of interest. The expressed gene product, e.g. protein may be harvested, collected or isolated either from the cells or from the culture medium or from both. It may then be further purified using known methods, e.g. filtration, column chromatography, etc, by methods generally known to the person skilled in the art.

The practice of this invention will employ, unless otherwise indicated, conventional techniques of immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art. See e.g. Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2^(nd) edition, 1989; Current Protocols in Molecular Biology, Ausubel F M, et al, eds, 1987; the series Methods in Enzymology (Academic Press, Inc.); PCR2: A Practical Approach, MacPherson M J, Hams B D, Taylor G R, eds, 1995; Antibodies: A Laboratory Manual, Harlow and Lane, eds, 1988. [0088] The invention is further explained in the following examples. The examples do not limit the invention in any way. They merely serve to clarify the invention.

In this document and in its claims, the verb “to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”.

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

DESCRIPTION OF THE FIGURES

FIG. 1. Genomic structure of genes that are screened for fragments that elevate the formation of colonies in the context of a stringent selection system.

FIG. 1 shows the six 3500 bp DNA stretches upstream from the transcription start site, as well as a ˜3500 bp DNA stretch coding region of the genes, encompassing the start of translation in the corresponding mRNA (dubbed Z) for each locus. The six upstream DNA stretches, containing only non-coding DNA, were dubbed A to F.

FIG. 2. Genomic sequences that induce more colonies than STAR elements in the context of stringent selection systems.

CHO-DG44 cells were transfected with 3 μg DNA of constructs as shown, using TTG-Zeo (in FIG. 2A) or pp 8-Zeo-EPP5 (in FIG. 2B) as selectable marker. For the negative control there is no sequence introduced as Element X. For the positive control STAR 7/67 is used as Element X at the 5′ end and STAR 7 is used as Element X at the 3′ end. The different stretches of DNA of FIG. 1 were used as element X as indicated. Selection was performed with 400 μg/ml Zeocin in the culture medium, which was added 24 hours after transfection. The culture medium consisted of HAMF12: DMEM=1:1+4.6% fetal bovine serum. After approximately two weeks the number of stably established colonies were counted.

FIG. 3. Rb1E, Rb1F and p15C induce equal or higher GFP expression levels than STAR elements.

d2EGFP expression levels were determined in stable colonies comprising DNA constructs described in FIG. 2. The relative fluorescence levels were taken as arbitrary units. The average d2EGFP expression levels for each construct are indicated with a line. The average d2EGFP expression of 615 induced by STARs 7/67/7 in the context of the TTG Zeo selection system is indicated with a bold line.

FIG. 4. Rb1E and p15C elements do not possess promoter activity.

The construct that contained STARs 7/67/7 and the β-actin promoter was modified in such a way that the β-actin promoter was replaced by either the Rb1E or p15C element. This created constructs that contained the Rb1E and p15C elements placed immediately upstream of the TTG Zeo d2EGFP cassette. As a control the constructs described in FIG. 2, that did harbor the β-actin promoter were used. We transfected the constructs to CHO-DG44 cells and measured the transient d2EGFP values. FIG. 4A shows the relative transient d2EGFP values. FIG. 4B shows the number of stable Zeocin resistant colonies.

FIG. 5. Rb1E and p15C elements are no enhancers.

Constructs as indicated in FIG. 5 were made. The Rb1E or p15C element was placed upstream of the SV40 minimal promoter and the combined TTG Zeo-d2EGFP gene. As control the β-actin promoter was placed upstream of the TTG Zeo-d2EGFP gene. In another control the SV40 minimal promoter was placed upstream of the TTG Zeo-d2EGFP gene. FIG. 5 shows the relative transient d2EGFP values of the different constructs.

FIG. 6. Rb1E and p15C elements do not in trans influence transcription of the endogenous Rb1 and p15 promoters.

FIG. 6 shows the relative Rb1/actin signal and the relative p15/actin signal as compared to wild type CHO-DG44. The ratio of the β-actin and the Rb1 mRNA level or the β-actin and the p15 mRNA level was determined by real time PCR. Four independent clones of each element were compared.

FIG. 7. Rb1E and p15C elements do not contain STAR activity.

FIG. 7A shows schematically what happens if an element has STAR activity or not. In short, the elements were placed between targeted LexA-HP1 repressors and the Zeocin selection gene. When the elements have no STAR activity, the HP1-mediated gene repression will silence the Zeocin selection marker gene. Subsequent addition of Zeocin to the culture medium will result in cell death. On the other hand, when an element does contain STAR activity, the HP1-mediated gene repression is not strong enough to silence the Zeocin selection marker. Subsequent addition of Zeocin to the culture medium will result in survival of these cells. FIG. 7B shows the results on survival of U2-OS cells, a human cell line (Human Osteosarcoma Cell line, ATCC HTB-96; described in Heldin, CH, et al. 1986, Nature 319: 4511-514).

FIGS. 8 and 9. The Rb1E element and the p15C element are a source of intergenic transcription.

To determine whether intergenic transcripts are associated with the Rb1E and p15C elements, four primer sets were designed for the Rb1E and p15C genomic elements. Using random hexamers cDNA was made from total RNA isolated from U2-OS cells. Using real time PCR it was determined whether there was an elevated level of RNA, transcribed across the tested region. The real time PCR reactions were performed on the cDNA, created from U2-OS cells. As control, the total RNA from which the cDNA was made, was used as sample for the real time PCR reaction. The difference in the respective signal levels in the RNA or cDNA samples was taken as measure for the level of intergenic transcripts. FIG. 8 shows the results for the Rb1E primers. FIG. 9 shows the results for the p15C primers.

FIG. 10. Copy numbers in clones that contain Rb1E or STAR elements.

FIG. 11. Rb1E and p15C are functional in the context of different promoters.

FIG. 11 shows the mean d2EGFP fluorescence level in Zeocin resistant colonies after transfection with the construct comprising the CMV promoter as schematically presented in FIG. 11. The number of colonies that was induced is indicated above the graph.

FIG. 12. Specific combinations of Rb1E and p15C to induce optimal colony numbers and protein expression levels.

In FIG. 12 the number of stable Zeocin resistant colonies is shown, whereby the colonies were transfected with a construct as schematically presented in FIG. 12 and wherein element X and element Y are indicated on the X-axis.

FIG. 13. Specific combinations of Rb1E and p15C to induce optimal colony numbers and protein expression levels.

In FIG. 13, the mean d2EGFP fluorescence level in the cells of FIG. 12 are shown.

FIG. 14. Testing of regions within Rb1E and p15C for highest activity.

FIG. 14 shows the number of stable Zeocin resistant colonies after transfection with a construct as schematically indicated in FIG. 14 and wherein Element X are either the full Rb1E or p15C elements or a part thereof.

FIG. 15. Testing of regions within Rb1E and p15C for highest activity.

In FIG. 15, the mean d2EGFP fluorescence level in the cells of FIG. 14 are shown.

FIG. 16. Testing of regions within Rb1E and Rb1F and a combination thereof for number of stable colonies. The following fragments were tested for number of stable colonies they produced: Rb1E: 1-3498, 1-2018, 1-1482, 1-1019, 1-479, 479-2018, 1019-2018, 1482-2018, 479-1482; Rb1F: 1-3424, 1-2425, 2425-3424; Rb1E/Rb1F: 2425-3424 (Rb1F)-1-2018 (Rb1E).

FIG. 17. Testing of regions within Rb1E and Rb1F and combination thereof for activity. The following fragments were tested for number of stable colonies they produced: Rb1E: 1-3498, 1-2018, 1-1482, 1-1019, 1-479, 479-2018, 1019-2018, 1482-2018, 479-1482; Rb1F: 1-3424, 1-2425, 2425-3424; Rb1E/Rb1F: 2425-3424 (Rb1F)-1-2018 (Rb1E).

FIG. 18. Rb1E and p15C induce high EPO protein expression levels.

EPO production levels are shown as achieved in cells that were stably transfected with the construct as schematically presented. The EPO reporter gene was under control of the β-actin promoter. As selectable marker the pp⁸Zeo^(EPP5) variant was used. FIG. 18A shows the specific EPO activity in pg per cell per day. FIG. 18B shows the volumetric EPO production in pg of EPO per day.

EXAMPLES 1. Example 1 Screening Specific Genomic Loci for Sequences that Convey Equal or More Stably Transfected Colonies than STAR Elements

When CHO-DG44 are transfected with a plasmid that harbor a stringent selectable marker such as the Zeocin resistance marker that is modified at its translation initiation codon, little or no colonies will emerge. This is specifically the case with a Zeocin resistance marker that has a TTG translation initiation codon and that is placed under the control of the human β-actin promoter (SEQ ID NO: 17) (See FIG. 2A). However, when STAR elements are placed to flank the entire expression cassette, many more colonies will emerge, typically in the range of 50-100 per transfection (see for instance FIG. 2A), when 400 μg/ml Zeocin is added to the CHO-DG44 culture medium. In general, the resulting clones convey high protein expression levels. Here, we attempted to identify genomic sequences that are able to induce at least as many CHO-DG44 colonies as with STAR elements under the same selection conditions. We therefore used the same Zeocin resistance marker as is used with STAR elements, TTG Zeo. The expression cassette was placed under control of the human β-actin promoter (FIG. 2A). Genomic loci of three human genes were chosen: Rb1, p15 and p73. Stretches of DNA of approximately the same length (˜3500 bp) were isolated by PCR using BAC clones as template. The numbers of these BAC clones were respectively RP11-136N2, RP11-478M20 and RP5-1092A11 (obtained from BacPAC Resources Center-BPRC) for Rb1, p15 and p73. For each locus we isolated and analyzed six ˜3500 bp DNA stretches upstream from the transcription start site, as well as a ˜3500 bp DNA stretch coding region of the genes, encompassing the start of translation in the corresponding mRNA (dubbed Z). The six upstream DNA stretches, containing only non-coding DNA, were dubbed A to F (FIG. 1). The specific sets of primers are given in table 1. The particular stretches of DNA were cloned to flank a construct encompassing the human β-actin promoter, the TTG Zeo resistance gene and the d2EGFP reporter gene. A short DNA sequence run was performed on the isolated DNA sequences to verify that we indeed isolated the intended sequence. This proved to be the case. As control constructs we took the same construct without any flanking DNA element and the same construct flanked with STARs 7 and 67 (disclosed in WO 2007/096399) upstream of the expression cassette and STAR 7 (disclosed in WO 2007/096399) downstream of the expression cassette (FIG. 2A).

TABLE 1 Primer sets for the isolation of genomic elements (5′ -> 3′ direction) SEQ SEQ ID ID forward primer NO reverse primer NO RB1 Z ggagcgtctgcagaatggtgacagg 18 agactctcgctctgttgccaggctg 39 RB1 A ctgaaggagtctcaaactgaagagag 19 acaaagagtctggtgggtgactgtg 40 RB1 B tgtttgcattcctgtagcccacaag 20 cgttctaaaaagccttccttcaaag 41 RB1 C gtgatgtaaatctttgcaattcttc 21 tcttaatggcttgatgagccacac 42 RB1 D tagtcttttgtatgtgataaatctc 22 taccattcaattctcccgtctgac 43 RB1 E gcccaccctaaatacttatacaggc 23 acaccccaggaacagaatcagtgc 44 RB1 F actatgtcatttttgctaacatgtaatgg 24 gctattcactcattcctgtagctgtctaat 45 P15 Z ggggactagtggagaaggtgcgaca 25 ccagggcttccagagagtgtcgttta 46 P15 A cctcttggtgggaaggtgtgttcataa 26 aagcctgcccaaagatgctaggacg 47 P15 B tcattgagcagtggtttgtagttctccttg 27 ttatgaacacaccttcccaccaagagg 48 P15 C ttagtctaaattagggatacacactcctcc 28 caatatcgtgaaaatggccatactg 49 P15 D atggaagatagtggaaccaacttggaaagc 29 tcaggggtacatgtgcaggtttgttacata 50 P15 E agctttagctactccagctttctgggtgt 30 tggaaaggtagtcttcaagcttggaaattc 51 P15 F tttcactacttcccctgtataacctccacg 31 aagatctgtgagagcagtgtggattccc 52 P73 Z gcaccacgtttgagcacctctggag 32 cagttttccagggggcactcagagc 53 P73 A tgtgatttggaataaaacctccctgaagagg 33 gcgggcgttagcgcctttttag 54 P73 B ccagacagctatgagcactcagtggact 34 cagggaggttttattccaaatcaca 55 P73 C aaatacatttaaaaatctggcagagccggg 35 tgatggagttggatcccagtgtttgg 56 P73 D atcaacgccaccgttcttccatgtc 36 cagtgccacctttctcttggttaggatttt 57 P73 E tactatcttgggatcattaatggctgcagg 37 caggcatccagttctgagctttctctct 58 P73 F cgcgaacagcctcagcttctgaatg 38 ggtgggaaactgctccttcactttgct 59

1.1 Results

We transfected the plasmids with the isolated DNA stretches from the Rb1, p15 and p73 loci to CHO-DG44 cells. The same amount of DNA (3 μg) of all constructs was transfected to CHO-DG44 cells with Lipofectamine 2000 (Invitrogen). Selection was performed with 400 μg/ml Zeocin in the culture medium, which was added 24 hours after transfection. The culture medium consisted of HAMF12: DMEM=1:1+4.6% fetal bovine serum. After approximately two weeks the number of stably established colonies were counted. As shown in FIG. 2A, transfection of the construct encompassing START/67/7 resulted in 105 stable colonies. Transfection of seven constructs containing DNA sequences from the p73 gave rise to hardly any colonies (FIG. 2A). In contrast, transfection of the constructs containing the DNA sequences from either the Rb1 or p15 loci gave a significant number of colonies. Specifically, the Rb1E, p15C and Rb1F sequences induced 247, 125 and 113 colonies respectively (FIG. 2). Since the Rb1E and p15C sequences induced ˜2.5 and ˜1.25 more colonies than STAR 7/67/7 elements respectively, we decided to focus on these sequences. Analysis of the sequences in databases such as blast revealed no known sequence motifs, promoter regions or repeats. No duplications of the sequences in the human genome were found either.

These experiments were performed with the TTG Zeo selection system that has been devised in the context of STAR elements. Recently, we developed a novel selection principle in which short peptides are placed upstream of a selectable marker, such as the Zeocin resistance marker. In essence, when this small peptide becomes longer, the translation machinery will have increasing difficulties to re-initiate at the translation initiation codon of the Zeocin mRNA. As a result higher levels of mRNA have to be produced in order to warrant enough translated, functional Zeocin resistance protein. This creates a stringent selection marker system, called ppZeo selection system. Here we tested whether the Rb1A to F elements as well as the p15C element are also able to induce more colonies with high d2EGFP expression levels when put in the context of the ppZeo selection system.

As selectable marker we used the pp⁸Zeo^(EPP5) variant (SEQ ID NO: 16). This variant harbors a small peptide of 8 amino acids and is placed upstream of a Zeocin selectable marker mutant that is more stringent than the wild type Zeocin marker. This mutant is created by Error Prone PCR (EPP). The pp⁸Zeo^(EPP5) variant provides slightly higher selection stringency than the TTG Zeo selectable marker.

We flanked the expression cassette (SEQ ID NO: 9) with the Rb1A-F and p15C sequences, as well with STARs 7/67/7 (SEQ ID NO: 10)(FIG. 2B). As shown in FIG. 2B, the STAR 7/67/7 combination induced 76 colonies, slightly less than with the TTG Zeo marker (FIG. 2A). This is in agreement with the notion that the pp8Zeo^(EPP5) marker is slightly more stringent than the TTG Zeo marker. Importantly, hardly any colony emerged when no elements at all were included in the construct. As with the TTG Zeo marker, the constructs containing the Rb1E, Rb1F and the p15C induced the most colonies in the context of the pp8Zeo^(EPP5) marker. Rb1E induced 163 colonies, Rb1F 124 colonies and P15C 69 colonies (FIG. 2B).

We conclude that some of the genomic DNA loci that we screened contain sequences that are able to induce an equal number or more colonies than STAR elements in the context of the two different, high stringency selection system.

2. Example 2 The Rb1E, Rb1F and p15C sequences induce equal or higher protein expression levels than STAR elements in the context of a stringent selection system.

Since the constructs that contain the Rb1E and p15C sequences also harbor the d2EGFP reporter gene, we were able to analyze the influence of the Rb1E and p15C DNA sequences on the d2EGFP expression levels.

2.1 Results

Between 12 and 24 independent colonies induced by the indicated constructs were isolated. Colonies were propagated before analysis by flow cytometric analysis (EPICS-XLM, Beckman-Coulter), 3 to 4 weeks after transfection. The fluorescence signal derived from d2EGFP (destabilized) is linear with the amount of available d2EGFP protein in a cell, and is thus a reliable indicator of the d2EGFP expression levels in the cell. In a single FACS analysis, fluorescence signals from a sample that contain up to 4000 cells are analyzed. One such sample of cells is taken from an independent, stably transfected cell colony. Since the signal will vary amongst the individual cells in the colony, the mean fluorescence level of the ˜4000 cells in the sample is taken as a measure for the d2EGFP expression level in the stably transfected cell colony.

As shown in FIG. 3, incorporation of the Rb1E, Rb1F and p15C sequences induced equal or slightly higher d2EGFP expression levels, as compared to the control construct with the STAR 7/67/7 elements. This was the case in the context of both the TTG Zeo and pp 8Zeo^(EPP5) markers. Overall, the d2EGFP expression values were highest with the Rb1E sequences, again, with both selection markers.

We conclude that the inclusion of the Rb1E, Rb1F or p15C sequences not only induces more colonies, but these colonies also display a higher d2EGFP expression level. This is tested in the context of a stringent selection system that is routinely used with STAR elements.

3. Example 3 The Rb1E and p15C sequences do not harbor promoter or enhancer activity, are no STAR elements, but are sources of intergenic transcription

Possible reasons for the ability of the Rb1E and p15C elements to induce a high number of colonies with high protein expression levels could be that these elements are promoters themselves. Alternatively, the elements could be STAR elements. We tested these possibilities experimentally.

3.1 Results

The construct that contained STARs 7/67/7 and the β-actin promoter was modified in such a way that the β-actin promoter was replaced by either the Rb1E or p15C element. This created constructs that contained the Rb1E and p15C elements placed immediately upstream of the TTG Zeo d2EGFP cassette. We compared these constructs with the constructs described in Example 2, that did harbor the β-actin promoter (FIG. 4). We transfected the constructs to CHO-DG44 cells and measured the transient d2EGFP values. As shown in FIG. 4A, the constructs with either the Rb1E or p15C element, but without β-actin promoter gave no d2EGFP signal at all. This indicates that the elements are no functional promoters. To further substantiate this notion we kept the transfected cells under Zeocin selection pressure. As shown in FIG. 4B, the constructs containing STAR elements, the Rb1E or p15C with the β-actin promoter induced 112, 275 and 154 colonies respectively. In contrast, the constructs with the Rb1E and p15C elements, but without β-actin promoter induced no colonies at all. Next, we tested whether the Rb1E or p15C elements might be enhancer elements. We tested this by placing the elements upstream of the SV40 minimal promoter and the combined TTG Zeo-d2EGFP gene. As control constructs we took the β-actin promoter upstream of the TTG Zeo-d2EGFP gene. We also placed the SV40 minimal promoter upstream of the TTG Zeo-d2EGFP gene. Finally, we placed the SV40 enhancer upstream of the SV40 minimal promoter. This is the natural occurring SV40 enhancer/promoter configuration. As shown in FIG. 5, only the constructs in which the β-actin promoter or the ‘complete’ SV40 enhancer/promoter combination was placed upstream of the reporter gene gave significant d2EGFP signals (arbitrarily put at 100). Neither construct with the SV40 minimal promoter gave any signal, indicating that the Rb1E nor p15C elements are no enhancers. Taken together these data show that the Rb1E and p15C elements are no functional promoters or enhancers.

We next tested whether stable transfection of the constructs harboring the Rb1E and p15C elements would in trans influence the endogenous CHO Rb1 or p15 expression. We devised a primer set that gave a positive mRNA signal, corresponding with the endogenous CHO Rb1 and p15 genes. The following primer sets were used:

(SEQ ID NO: 80) P15 Forward: GGAGCAGAACCCAACTGCGC (SEQ ID NO: 81) P15 Reverse: CCAGGCGTCACACACATCCAG (SEQ ID NO: 82) RB1 Forward: GTGACAGAGTGCTCAAAAGAAGTGCTG (SEQ ID NO: 83) RB1 Reverse: GGACTCCGCTGGGAGATGTTTACTC

Subsequently, we measured the ratio of the β-actin and the Rb1 mRNA level or the β-actin and the p15 mRNA level, by real time PCR. We compared these ratios in CHO-DG44 versus Rb1E or p15C transfected colonies. We compared four independent clones of each element. In FIG. 6 we show the result for one clone. We found that transfection of a construct containing either the Rb1E or p15C element did not influence the ratio between the β-actin and respective endogenous Rb1 or p15 genes. This was the case in all four independent clones.

We conclude that transfection of the Rb1E or p15C elements do not have a positive or negative effect on the expression of the respective endogenous genes.

We also tested whether the Rb1E or p15C elements harbor STAR activity. This can be directly tested by placing the elements between targeted LexA-HP1 repressors and the Zeocin selection gene. When the elements have no STAR activity, the HP1-mediated gene repression will silence the Zeocin selection marker gene. Subsequent addition of Zeocin to the culture medium will result in cell death. On the other hand, when an element does contain STAR activity, the HP1-mediated gene repression is not strong enough to silence the Zeocin selection marker. Subsequent addition of Zeocin to the culture medium will result in survival of these cells. These experiments were performed in U2-OS cells, as was the original screen to identify and isolate STAR elements (Kwaks et al., 2003, Nature Biotech. 21: 553-558). As shown in FIG. 7, placing STAR 7 between the LexA-HP1 binding sites and the Zeocin marker gene does indeed result in cell survival and resulting, fast growing colonies. As shown in FIG. 7, neither the testing of the full-length Rb1E, Rb1F and p15C elements or shorter fragments resulted in the emergence of colonies. The smaller fragments corresponded with the fragments that were also tested for their ability to induce a high number of colonies with high protein expression levels (see example 5; FIGS. 14 and 15). We also tested the Rb1E/Rb1F combination (2425-3224 (Rb1F)-1-2018 (Rb1E)) for STAR activity and found no such activity (FIG. 7). We conclude from these results that neither Rb1E, Rb1F nor P15C contain STAR activity.

Finally, we tested the possibility that the Rb1E or p15C elements as sources of intergenic transcription. Rb1E and p15C harbor a striking ability to induce many colonies with high protein expression levels in the context of a stringent selection system. As shown above, they do not contain promoter, enhancer of STAR activity. We therefore tested whether they are regions in which intergenic transcription takes place.

To determine whether intergenic transcripts are associated with the Rb1E and p15C elements, we designed five primer sets for the Rb1E and p15C genomic elements.

TABLE 2 Primer sets for the performance of real time  PCR and detection intergenic transcription  (5′ → 3′ direction). SEQ ID primer Sequence NO P15C 50 F GATACACACTCCTCCCTGAGCTCTAGAC 60 P15C 232 R AATGAGAGAGGTTGGGATCATGGTC 61 P15C 537 F GTCCTAACATGGCCTATACAGCTCTACAAC 62 P15C 691 R CAGAAGAAACTGCATGTGGCAAGC 63 P15C 1468 F TCAACCTCTGCCTCCTGGGTTC 64 P15C 1613 R TTCAAGACCAGCCTGACCAACATG 65 P15C 2317 F TTGTGTGAAACGGGTAGGTTGAGC 66 P15C 2497 R GCCAATATGGTGAAACCCCATCTC 67 P15C 3133 F CTCTGTTTTGGTACCAGTACCATGCTG 68 P15C 3274 R ATATGGAACCAAAAAGGAGCCCG 69 RB1 E 134-F AAGCTTCCTGACTTCAGCCTAAAGATTC 70 RB1 E 292-R CTTACCTGACATTTCTGTCATCTTCCTCTTC 71 RB1 E 941-F CTCATACGCATATCATGTGGACAAAGTG 72 RB1 E 1112-R GGCAACAGAGCGAGACTCAGTCTC 73 RB1 E 1714-F ATCCCACTGAATTACTGAGAGGATTGATC 74 RB1 E 1886-R CCATGTCCTTGTGTTGAGCTCTCTG 75 RB1 E 2561-F ATAGCTAAACTGTCTTCTCAGGAGAGGAGC 76 RB1 E 2677-R CTCTGCTTGGCATCTACCTCCAAAC 77 RB1 E 3374-F GAACTTGCACTTGTCCCACATCCAG 78 RB1 E 3508-R CAGGAACAGAATCAGTGCTTTTTCCTC 79 F = forward primer; R = reverse primer

Using random hexamers we made cDNA from total RNA, isolated from U2-OS cells. We selected this human cell line to assess whether there were endogenous intergenic transcripts associated with the indicated genomic loci. With real time PCR we determined whether there was an elevated level of RNA, transcribed across the tested region. The real time PCR reactions were performed on the cDNA, created from U2-OS cells. As control, the total RNA from which the cDNA was made, was used as sample for the real time PCR reaction. Contamination with genomic DNA in the RNA sample would also give a background signal. The difference in the respective signal levels in the RNA or cDNA samples was taken as measure for the level of intergenic transcripts. As shown in FIGS. 8 and 9, we found with three out of five primer sets positive Rb1E signals and with four out of five primer sets p15C signals when using cDNA and RNA isolated from U2-OS cells (first columns in respectively FIGS. 8 and 9). The indicated factor is the difference in signal level in the cDNA sample versus the RNA sample. These data indicate that intergenic transcripts are associated with the Rb1E and p15C loci.

We next tested whether such positive signals could also be detected in CHO-DG44 colonies that were induced by constructs containing either the human Rb1E or p15C elements. As source for the RNA/cDNA we took the same four colonies in which we tested whether the elements had an in trans influence on the expression of the endogenous CHO Rb1 or p15 promoters (FIG. 5). As negative controls we included RNA or cDNA from four clones that were transfected with another construct. Hence RNA/cDNA from cells transfected with the p15C element served as negative control in the test for intergenic Rb1E transcripts (FIG. 8) and vice versa (FIG. 9). As shown in FIG. 8, there was substantial intergenic transcription at the same three of the five different locations within the Rb1E element as in U2-OS cells (second columns in FIG. 8). Importantly, no such positive signal was detected when p15C-transfected clones were taken as source for the RNA/cDNA samples (third columns in FIG. 8). It should be noted the absolute amount of detected transcripts was higher in the Rb1E transfected cells than in U2-OS cells, probably due to the fact that multiple copies harboring the Rb1E element are transfected, while the U2-OS cells have only two endogenous copies. However, the ratios between the cDNA and RNA signals were the same and these are indicated in FIGS. 8 and 9.

As shown in FIG. 9, there was also substantial intergenic transcription at the same four of the five different locations within the p15C element as in U2-OS cells (second columns in FIG. 9). Importantly, no such positive signal was detected when Rb1E-transfected clones were taken as source for the RNA/cDNA samples (third columns in FIG. 9).

As overall conclusion for this example we take it that the ability of Rb1E and p15C elements to induce a high number of colonies with high protein expression levels is not due to endogenous promoter, enhancer activity or STAR activity. Instead they appear to contain regions that are associated with intergenic transcriptions. A possibility is that due to this intergenic transcription the locus signifies an open chromatin structure that is pivotal enabling high transcription levels from the downstream promoter.

4. Example 4 Rb1E Induced High Colony Number and d2EGFP Values are not Due to an Increased Number of Plasmid Copies

The Rb1E element induces more colonies than STAR elements and with at least equally high d2EGFP values. One possibility might be that inclusion of the Rb1E element might result in stable colonies that have more copies of the plasmid incorporated. We tested this by directly determining the copy numbers of the respective plasmid in a seven independently isolated stable colonies.

4.1 Results

We isolated DNA from seven clones that were transfected with either STAR 7/67/7 or Rb1E elements. The average d2EGFP values in the seven STAR-induced colonies was 156, and in the seven Rb1E-induced colonies 299. As shown in FIG. 10, the average copy number in STAR-induced colonies was 79, whereas the average copy number in Rb1E-induced colonies was 17. It therefore appears that the high d2EGFP values induced by Rb1E are not due to an increased copy number, but that, instead more d2EGFP is produced per copy.

We also placed the Rb1E and p15C sequences around an expression cassette harboring the CMV promoter, the TTG Zeo selectable marker and the d2EGFP reporter gene. The constructs containing the Rb1E or p15C induced 176 and 107 colonies, as compared to the 152 colonies induced by the STAR 7/67/7 combination (FIG. 11). Up to 24 independent colonies were isolated, propagated and d2EGFP was analyzed. As shown in FIG. 4, the Rb1E and p15C sequences induced average d2EGFP expression levels of 957 and 825 respectively, as compared to the average d2EGFP expression of 862 induced by STARs 7/67/7 (FIG. 11).

5. Example 5 Specific Combinations of Rb1E and p15C Sequences and Localization of Highest Activity within the DNA Stretches

We tested the effects of employing different combinations of the Rb1E and p15C sequences. Also, we tested different portions of the elements to analyze whether there is a localized activity within these sequences.

5.1 Results

As shown in FIG. 12, we made constructs in which the Rb1E or p15C element was place only upstream or downstream, as well as flanking the entire expression cassette. Furthermore, we made constructs in which the Rb1E element was placed upstream and the p15C element downstream of the expression cassette. Vice versa, we placed the p15C upstream and the Rb1E element downstream of the expression cassette. FIG. 12 shows that when the Rb1E was placed downstream as single element, colony numbers were significantly higher than when the single Rb1E element was placed upstream of the expression cassette. However, most colonies were induced when two Rb1E elements were used to flank the entire expression cassette. In contrast, no such distinction was found with the p15C element (FIG. 12). Finally, when the Rb1E element was placed downstream and the p15C upstream of the expression cassette, more colonies were induced than when the order of the elements was reversed (FIG. 12). This again shows the dominance of the downstream position for the Rb1E element in a construct.

When we analyzed the d2EGFP expression levels in the respective clones, we found no major differences in the average d2EGFP expression levels (FIG. 13). Although the differences were not much, the highest d2EGFP levels were found with the Rb1E elements on both sides and with the p15C-Rb1E combination. We conclude from these data that both in terms of inducing a high number of colonies and of protein expression levels it is beneficial that two elements are used instead of one.

Next we analyzed different portions of the Rb1E and p15C elements. As shown in FIG. 14, the 1-3498 long by of the Rb1E element was compared to the 1-2018 bp and the 1482-3498 bp region of Rb1E. Likewise, the 1-3352 long by of the p15C element was compared to the 1-1500 bp and 822-3352 bp region of p15C. The most obvious result was that the 1450-3500 bp region of Rb1E did not induce a significant number of colonies, as compared to the full-length sequence and the 1-2018 bp region (FIG. 14). In fact, the 1-2018 bp region appears to harbor most of the ability of Rb1E to induce a high number of colonies in CHO-DG44. In contrast, no such striking result was found with the p15C element. Although the 1-1482 region gave less colonies than the 850-3352 bp region, this difference was less outspoken than with the Rb1E element (FIG. 14). When we analyzed the d2EGFP expression levels in the clones described above, we noted that there were no major differences between the full-length elements and the specific portions (FIG. 15). We conclude from these data that the best configuration of the Rb1E and P15C elements is when used as homologous pair to flank the expression cassette. Only the Rb1E element can be delineated into specific parts, particularly in terms of its ability to induce high numbers of colonies.

We further delineated the Rb1E (1-2018 bp) element to define the minimal sequence that gave both the highest number of colonies and the highest d2EGFP values. As shown in FIG. 16, reducing the Rb1E 1-2018 fragment to 1-1482 bp reduced the number of colonies significantly. Furthermore, the 1-1019 bp fragment gave very little colonies and 1-479 hardly any. Also a small reduction of the 1-2018 fragment from the other side (479-2018 bp) had a dramatic impact on the number of induced colonies. It appears that for optimal colony formation the entire 1-2018 bp region is essential; further shortening of this fragment from either side immediately makes the fragment less effective in inducing a large number of colonies. We next considered the Rb1F fragment. As described in Example 1 (FIG. 2), the Rb1F fragment also induced a significant number of stable colonies, although less than the Rb1E fragment. However, initially, these fragments are merely chosen on the basis of their sequential order in the genomic locus of Rb1. Simply, 3424 bp stretches of genomic Rb1 DNA are isolated and tested. It is well possible that some of the activity we define in the Rb1E fragment overlaps with the joining fragment, Rb1F. We therefore tested which parts of the Rb1F encompassed the highest activity and whether this is adjacent to the Rb1E fragment. We divided the Rb1F fragment into two fragments, 1-2425 and 2425-3424 the last being adjacent to the Rb1E fragment. As shown in FIG. 16, the 2425-3424 bp fragment induced the highest number of colonies, almost as many as the entire, 1-3424 bp fragment. We therefore joined the two fragments, Rb1F (2425-3424) with Rb1E (1-2018) and tested the activity of this combination. As shown in FIG. 16, the combination induced the highest number of colonies, even slightly more than the Rb1E (1-3498) fragment. We conclude that this specific combination encompasses the highest activity of the tested Rb1 locus to induce a high number of stable colonies.

When we analyzed the d2EGFP values in the described fragments, we found the following picture (FIG. 17). Of Rb1E, the entire Rb1E (1-3498) and the Rb1E (1-2018) fragments gave the highest d2EGFP values, as shown above. Of Rb1F, the entire Rb1F (1-3424) and Rb1F (2425-3424) fragments gave highest d2EGFP values (FIG. 17). However, highest d2EGFP values were achieved with the combined Rb1F (2425-3424)/Rb1E (1-2018) fragment. As with the induced number of colonies, the combined element is apparently the best combination, also for inducing high protein expression levels.

6. Example 6 The Rb1E and p15C Elements Induce High EPO Protein Expression Levels 6.1 Results

As shown in FIG. 18, we placed the Rb1E or p15C elements upstream of the β-actin promoter, driving the human erythropoietin (EPO) reporter gene. As selectable marker we used the pp⁸Zeo^(EPP5) variant (SEQ ID NO: 16). This variant harbors a small peptide of 8 amino acids and is placed upstream of a Zeocin selectable marker mutant that is more stringent than the wild type Zeocin marker. This mutant is created by Error Prone PCR (EPP) and has been described previously (U.S. provisional application 61/187,022). The pp⁸Zeo^(EPP5) variant provides slightly higher selection stringency than the TTG Zeo selectable marker.

We found that both the Rb1E and p15C elements were able to induce large numbers of stable EPO producing colonies (50 and 46 respectively), as compared to the 22 colonies induced by the STAR 7/67/7 combination. When specific EPO production levels were analyzed in the clones, we found that the Rb1E and p15C elements induced similar EPO expression levels as the STAR 7/67/7 combination (FIG. 18A). When also cell growth was taken into account, allowing an assessment of the volumetric EPO production, we found that the Rb1E element gave slightly better values than either the STAR 7/67/7 or the p15C combinations (FIG. 18B). We conclude that the Rb1E and p15C elements are able to induce a higher number of EPO producing colonies with similar EPO expression. This is the same conclusion as with d2EFP as reporter gene. 

1. A nucleic acid fragment comprising: (a) between 1,000 and 15,000 consecutive nucleotides of a genomic region that is present upstream of the translation initiation site of a vertebrate Rb1 gene; or, (b) at least 1500 consecutive nucleotides from a genomic region that is present from 10.5 to 7 kilobases upstream of the translation initiation site of a vertebrate p15 gene; wherein the fragment, when directly flanking an expression cassette having the nucleotide sequence of SEQ ID NO: 9, both up- and downstream of the expression cassette, produces at least 50% of number of colonies obtained with the same expression cassette when flanked with STARs 7 and 67 upstream of the expression cassette and STAR 7 downstream of the expression cassette SEQ ID NO: 10, when tested under the conditions of Example
 1. 2. A nucleic acid fragment according to claim 1, wherein the fragment has at least 80% nucleotide sequence identity over its entire length with at least 1000 consecutive nucleotides of at least one of SEQ ID NO's: 1-4 or
 8. 3. A nucleic acid fragment according to claim 1, wherein the fragment is from a human, mouse, rat, hamster, bovine, chicken, dog, cavia, pig or rabbit genome.
 4. A nucleic acid fragment according to claim 1, wherein the nucleic acid fragment is a fragment having at least 80% nucleotide sequence identity over its entire length with a fragment comprising: (a) nucleotide residues 1-1019, 1-1482, 1-2018, 1-3498, 479-2018 or 479-1482 of SEQ ID NO: 5; (b) nucleotide residues 1-2448, 1-3424 or 2425-3424 of SEQ ID NO: 6; (c) nucleotide residues 1-3064, 1-2500 or 1-2000 of SEQ ID NO: 7; and, (d) nucleotide residues 1-1500, 822-3352 or 1-3352 of SEQ ID NO:
 8. 5. A nucleic acid construct comprising a nucleic acid fragment according to claim 1, wherein the fragment is linked to at least one nucleotide that does not naturally occur immediately adjacent to the fragment in the genome from which the fragment is derived.
 6. A nucleic acid construct according to claim 5, wherein the nucleic acid fragment is linked to an expression cassette comprising a promoter operably linked to a nucleotide sequence encoding a gene product of interest.
 7. A nucleic acid construct according to claim 6, wherein the construct comprises a nucleic acid fragment upstream and downstream of the expression cassette comprising: (a) between 1,000 and 15,000 consecutive nucleotides of a genomic region that is present upstream of the translation initiation site of a vertebrate Rb 1 gene; or, (b) at least 1500 consecutive nucleotides from a genomic region that is present from 10.5 to 7 kilobases upstream of the translation initiation site of a vertebrate p15 gene; wherein the fragment, when directly flanking an expression cassette having the nucleotide sequence of SEQ ID NO: 9, both up- and downstream of the expression cassette, produces at least 50% of number of colonies obtained with the same expression cassette when flanked with STARs 7 and 67 upstream of the expression cassette and STAR 7 downstream of the expression cassette SEQ ID NO: 10, when tested under the conditions of Example
 1. 8. A nucleic acid construct according to claim 7, wherein the nucleic acid fragment upstream of the expression cassette is different from the nucleic acid fragment downstream of the expression cassette.
 9. A nucleic acid construct according to claim 6, wherein the expression cassette further comprises a nucleotide sequence encoding a selectable marker functional in a eukaryotic host cell.
 10. A nucleic acid construct according to claim 9, wherein the selectable marker provides resistance against lethal or growth-inhibitory effects of a selection agent selected from the group consisting of zeocin, puromycin, blasticidin, hygromycin, neomycin, methotrexate, methionine sulphoximine and kanamycin.
 11. A nucleic acid construct according to claim 9, wherein the nucleotide sequence encoding the selectable marker is a least one of: a) a nucleotide sequence having a mutation in the startcodon that decreases the translation initiation efficiency of the selectable marker polypeptide in a eukaryotic host cell; b) a nucleotide sequence that is part of a multicistronic transcription unit comprising i) the nucleotide sequence encoding the selectable marker; and, ii) a functional open reading frame comprising in a 5′ to 3′ direction a translation initiation codon, at least one amino acid codon and a translation stop codon; wherein the stop codon of functional open reading frame is present between 0 and 250 nucleotides upstream of the separate translation initiation codon of the nucleotide sequence encoding the selectable marker, and wherein the sequence separating the stop codon of functional open reading frame and the separate translation initiation codon of the nucleotide sequence encoding the selectable marker is devoid of translation initiation codons; and, c) a nucleotide sequence encoding a selectable marker polypeptide comprising a mutation encoding at least one amino acid change that reduces the activity of the selectable marker polypeptide compared to its wild-type counterpart.
 12. An nucleic acid construct according to claim 9, wherein the nucleotide sequence encoding a selectable marker and the nucleotide sequence encoding a gene product of interest are comprised in a single multicistronic transcription unit, wherein the multicistronic transcription unit is operably linked to the promoter and to a transcription termination sequence downstream of the multicistronic transcription unit.
 13. A nucleic acid construct according to claim 6, wherein the promoter is a β-actin promoter, a CMV promoter, an SV40 promoter, an ubiquitin C promoter or an EF1-alpha promoter.
 14. An expression vector comprising a nucleic acid fragment according to claim
 1. 15. A host cell comprising an expression vector according to claim
 14. 16. A host cell according to claim 15, wherein the host cell is a plant cell or a mammalian cell.
 17. A host cell according to claim 15 wherein the host cell is of a cell line.
 18. A host cell according to claim 16, wherein the cell line is selected from the group consisting of a U-2 OS osteosarcoma, CHO, CHO-K1, CHO-DG44, CHO-DG44-S, PER.C6, HEK 293, HuNS-1 myeloma, WERI-Rb-1 retinoblastoma, BHK, Vero, non-secreting mouse myeloma Sp2/0-Ag 14, non-secreting mouse myeloma NSO and NCI-H295R adrenal gland carcinoma cell line.
 19. A method of generating a host cell for expression of a gene product of interest, wherein the method comprises the steps of: (a) introducing into a plurality of host cells an expression vector according to claim 14; (b) culturing the plurality of host cells obtained in a) under conditions selecting for expression of the selectable marker polypeptide; and, (c) selecting at least one host cell expressing the selectable marker polypeptide for expression of the gene product of interest.
 20. A method of expressing a gene product of interest, comprising culturing a host cell according to claim 15, and expressing the gene product of interest from the nucleic acid construct.
 21. A method according to claim 20, further comprising recovery of recovering the gene product of interest. 